Combination of opioids and anticancer drugs for cancer treatment

ABSTRACT

The invention relates to novel strategies for the treatment of cancer patients based on a combination of an opioid receptor agonist and an anticancer compound.

The invention relates to novel strategies for the treatment of cancerpatients based on a combination of an opioid receptor agonist and ananticancer compound.

BACKGROUND OF THE INVENTION

Cancer can be defined as an abnormal growth of tissue characterized by aloss of cellular differentiation. This term encompasses a large group ofdiseases in which there is an invasive spread of undifferentiated cellsfrom a primary site to other parts of the body where furtherundifferentiated cellular replication occurs, which eventuallyinterferes the normal functioning of tissues and organs.

Cancers are primarily an environmental disease with 90-95% of casesattributed to environmental factors and 5-10% due to genetics.Environmental, as used by cancer researchers, means any cause that isnot inherited genetically, not merely pollution. Common environmentalfactors that contribute to cancer death include tobacco (25-30%), dietand obesity (30-35%), infections (15-20%), radiation (both ionizing andnon-ionizing, up to 10%), stress, lack of physical activity, andenvironmental pollutants.

With more than 3 million new cases and 1.7 million deaths each year,cancer represents the second most important cause of death and morbidityin Europe. On a global scale, cancer accounted for 7.4 million deaths(around 13% of the total) in 2004.

Although more than 40% of cancer deaths can be prevented, cancer is aleading cause of death, causing 20% of the total in the European Region.Noticeably, Europe comprising only one eighth of the total worldpopulation but has around one quarter of the global total of cancercases: some 3.2 million new patients per year.

The most common forms of cancer were prostate, colorectal, breast,leukemia and lung cancer. The risk of getting cancer before the age of75 years is 26.5%, or around one in four. However, because thepopulation of Europe is ageing, the rate of new cases of cancer is alsoexpected to increase.

Each cancer is characterized by the site, nature, and clinical cause ofundifferentiated cellular proliferation, whereby the underlyingmechanism for the initiation of cancer is not completely understood.

Cancer is usually treated with chemotherapy, radiation therapy andsurgery. Chemotherapy in addition to surgery has proven useful in anumber of different cancer types including: breast cancer, colorectalcancer, pancreatic cancer, osteogenic sarcoma, testicular cancer,ovarian cancer, and certain lung cancers. Radiation therapy involves theuse of ionizing radiation in an attempt to either cure or improve thesymptoms of cancer. It is used in about half of all cases and theradiation can be from either internal sources in the form ofbrachytherapy or external sources. Radiation is typically used inaddition to surgery and or chemotherapy but for certain types of cancersuch as early head and neck cancer may be used alone. For painful bonemetastasis it has been found to be effective in about 70% of people.

Despite the numerous therapeutic strategies there are still tumourswhich cannot be effectively treated with the current treatment options.In addition, the effectiveness of radiation- and chemotherapy is oftenlimited by toxicity to other tissues in the body.

Furthermore, anticancer therapies are frequently ineffective due toresistance of the tumour cells to radio- and/or chemotherapy.

Thus in oncology there is a great need for novel strategies, whichrender cancer treatments more effective. In particular, it is theobjective of the present invention to provide novel means for treatingcancer patients.

SUMMARY OF THE INVENTION

This objective is solved by using a combination of opioid receptoragonists and anticancer drugs for the treatment of cancer wherein thiscombination is given in a specific administration scheme according toclaim 1 of the invention.

In a first aspect the invention relates to a combination of an opioidreceptor agonist and at least one anticancer agent for use in thetreatment of cancer, wherein

-   -   (a) said opioid receptor agonist is administered to a patient in        one or more doses to establish a therapeutically effective        plasma level for a period of at least one week, and    -   (b) at least one anticancer agent selected from the group        consisting of chemotherapeutical agents, cytotoxic agents,        cytostatic agents, immunotoxic agents and/or radiotherapy is        administered to establish a period with a therapeutically        effective plasma level, and    -   (c) said periods of a) and b) overlap.

This combination therapy is based on the unexpected finding that opioidreceptor agonists together with anticancer agents kill cancer cells moreeffectively. Furthermore, the inventors could show that the interactionbetween opioid receptor agonists and anticancer agents represents aself-reinforcing feedback loop as illustrated by FIG. 26. In the firstpath of this loop opioid receptor agonists enhance the cellular uptakeand inhibit the efflux of anticancer drugs. In the second path of saidloop the accumulating anticancer drugs lead to an increased expressionof opioid receptors on the surface of the cancer cell. Hence, both theopioid receptor agonist and the anticancer agent can exert theircytotoxic potential to a higher extent.

Furthermore the invention is based on the unexpected finding that theamount of opioid receptor expressed on the cell surface of cancer cellsis varying among the different cancer types and also exhibitinginter-individual differences and that this surface-associated opioidreceptor expression can be increased by anticancer agents. For exampledoxorubicin, idarubicin, epirubicin, daunorubicin, carboplatin,oxaliplatin, cisplatin, etoposide, methotrexate, cytarabine, teniposide,rituximab fludarabine, are able to induce an increase of the number ofopioid receptors which are expressed on the cell surface of cancercells.

By extensive in vitro and in vivo experiments it could be shown thatdifferent cancer types can be subjected to the combination therapy ofthe invention. Furthermore also different anticancer drugs and differentopioids proved to be active in the above described feedback loop.

Therefore, the combination therapy of opioid receptor agonist andanticancer drug according to the invention can improve cancer therapy byone or more of the following ways:

-   -   Due to the upregulation of opioid receptors, former opioid        insensitive cancer types could be subjected to an opioid        receptor agonist therapy.    -   Due to the opioid-receptor-agonist-induced intracellular        accumulation (by either an increased uptake of anticancer drugs        or a reduced efflux or a combination of both) of anticancer        drugs the efficacy of the treatment is enhanced.    -   This could lead to therapy of cancer types which are        non-treatable or not effectively treatable by conventional        therapeutic anticancer approaches.    -   Furthermore, this might allow a dose reduction for the        anticancer drugs enhancing the safety and patient compliance of        the chemotherapy.    -   Finally, also resistant cancer cells could be re-sensitized for        an anticancer treatment.    -   In addition, the numerous opioids and numerous anticancer drugs        on the market open up the way for new drug combinations which        might represent improved treatment due to increased efficacy        and/or safety.

In the context of the present invention the term “opioid receptoragonist” is defined as a chemical heterogeneous group of natural,synthetic or semi-synthetic substances, working agonistic at the sametype of receptor, the so called opioid receptor. According to theanalgesia and side effect profile five types of opioid receptors, theμ-receptor (ligand=morphine), the K[kappa]-receptor (ligand=ketazocine),the delta-receptor (ligand=deltorphine II), the σ[sigma]-receptor(ligand=SKF 10081), as well as the later-identified ORL1-receptor(ligand=nociceptin) are known. Corresponding to other receptor systems,binding studies as well as functional investigations indicate thatsubtypes of opioid receptors exist. Within the μ- and δ^(i)-receptortype 2 subtypes, the μ-1 and μ-2 and δ-1 and δ-2 have been described.The κ-receptor contains an additional κ-3 subtype. Especially in regardsto the μ-opioid receptor its two subtypes are included in thisinvention.

The term “opioid receptor agonist” as used herein comprises fullagonists as well as mixed agonists/antagonists or partial agonists suchas buprenorphine.

The group of opioids includes natural opiates such as alkaloids likemorphine or dihydrocodeine, as well as semi-synthetic opiates, derivedfrom the natural opiates (e.g. hydromorphone or oxycodone), or fullysynthetic opioids, such as fentanyl or buprenorphine. It also includesendogenous opioid peptides, which may be produced naturally in the bodyas endorphins, dynorphins or enkephalins but which can also besynthesized.

As used herein the term “anticancer drug” encompasses all chemical orphysical interventions that are used for the treatment of cancer. Ittherefore includes chemotherapeutical agents such as cytotoxic agents orimmunotoxic agents but also radioactively labelled antibodies, peptidesand chemical substances, which might emit alpha, beta and gamma rays aswell as electrons. The radiotherapy further includes photons ofsufficiently high energy, charged particles such as electrons,positrons, muons, protons, alpha particles, and heavy atomic nuclei fromaccelerators, but also neutrons and gamma rays.

The term “therapeutically effective plasma level” is defined as a plasmalevel that is between the plasma level of the drug that causes a lethaleffect and the minimum plasma level that causes a therapeutic effect. Inthe context of the invention the therapeutic effect of the opioidreceptor agonist is given by the increase in cellular uptake and/or theinhibition of the cellular efflux of the co-administered anticancer drugand/or the induction of cell death by e.g. apoptosis, necrosis, mitoticcatastrophe and autophagy. In the context of the invention thetherapeutic effect of the anticancer drug is given by its ability tokill cancer cells and/or to induce the opioid receptor expression on thecancer cells.

There are two ways to look at the results of cancer treatment. Onecommon way is the measurement of cell death (increasing data means morecells are dead). The other way is to measure the viability of cells(decreasing data means that less living cells are present or have losttheir proliferation potential).

As used in the context of the present invention the words “treat,”“treating” or “treatment” refer to using the combination of the presentinvention or any composition comprising them to either prophylacticallyprevent a cancer, or to mitigate, ameliorate or stop cancer. Theyencompass either curing or healing as well as mitigation, remission orprevention, unless otherwise explicitly mentioned. Also, as used herein,the word “patient” refers to a mammal, including a human.

According to the invention the treatment specifically refers to theinhibition of cancer cell proliferation and/or growth. This activity caninclude e.g. cytostatic or cytotoxic activity as well arresting growthof cells and/or tumours. Cancer cell proliferation is the result of theinhibition of cell division. In particular opioid receptor agonistsinduce cell death in tumours. Cell death in the context of the inventionincludes all types of cells death. This can include necrotic as well asapoptotic cell death or autophagy. In one embodiment of the inventionthe cell death is induced by the activation of the caspases-dependent orcaspases-independent pathway. However, opioid receptor agonists caninduce cell death via various pathways. In a preferred embodiment of theinvention, opioid receptor agonists induce apoptosis in cancer cells.

As used herein, the term “cancer” which is synonymously used to the term“neoplasm” refers to diseases in which abnormal cells divide withoutcontrol and are able to invade other tissues. Cancer cells can spread toother parts of the body through the blood and lymph systems.

-   -   The terms “conventional therapy” and “conventional therapy        regimen” in the context of the present invention are defined as        the treatment programs (concerning dose, iteration-time and        duration) which are recommended as therapeutic guidelines of        associations, federations like Deutsche Krebshilfe, Deutsche        Krebsgesellschaft, Nationacl Cancer Institute (NCI), National        Comprehensive Cancer Network (NCCN) and respective health or        cancer organizations which could be private, non-governmental or        federal organizations. This also includes the treatment programs        for a (preferably single) anticancer agent as prescribed by the        manufacturer or distributor of the anticancer agent which are        disclosed in the respective instruction leaflets of the        anticancer agents.

The term “conventional therapy time” in the context of the presentinvention is defined as the time in a conventional therapy where ananticancer agent is applied to a patient without an opioid receptoragonist according the invention. The therapy time starts with the firstapplication of the anticancer agent, and may include iteration-periodswhich are specific for cancer and anticancer agent (for exampleapplication of a dose two times a day for a week than a pause of threeweeks and then again application of a dose two times a day for a weekfollowed by a pause of three weeks), up to time point, at which theanticancer agent is below the therapeutic plasma level of the patient.

Cancer and its different types in the context of the present inventioncan be classified by the ICD-O Standard which is a specialisedclassification of the ICD-10 Standard Classes C00-C97 and D00-D36.Alternatively, the classification of Boecker et al. 2008 in chapter 6(Pathologie, Elsevier, Urban & Fischer, p 167-218) can be used.

In a further embodiment of the invention said opioid receptor agonist iscapable of inhibiting cell proliferation.

In one embodiment of the invention said anticancer agent and said opioidreceptor agonist are administered simultaneously or successively.

In a preferred embodiment of the invention the periods of thetherapeutically effective plasma levels of the opioid receptor agonistand the anticancer agent, respectively overlap predominantly.

In a further preferred embodiment of the invention the period of thetherapeutically effective plasma levels of the anticancer agent iscompletely within the respective period of the opioid receptor agonist.

When administering two or more anticancer agents the respective periodfor which a partial, predominant or complete overlap is claimed, isgiven by the combined periods of the two or more anticancer agents.

In a further embodiment of the invention the opioid receptor agonist isgiven in a way that the patient develops a habituation against saidopioid receptor agonist. It is thus preferable to wait with theanticancer treatment until the habituation period has begun or evenreaches a plateau. The habituation can be a result of a decreased drugefficacy and/or a decrease in side effects such as respiratorydepression. Side effects of opioid receptor agonist are hypotension,respiratory depression, vomiting, constipation, dizziness, sedation,euphoria and cardiac effects. This side effects have to be taken inaccount for determine the therapy scheme with the opioid receptoragonist and the cancer agent. This means that the opioid receptoragonist is given at a starting dose on a very low level i.e. 1% of thetherapeutic dose and then increasing the dose depending to theguidelines of the opioid receptor agonist known by a skilled person andpublished by the manufacture or distributor of the opioid receptoragonist in an adequate time up to the therapeutic level which isrequired for the combination of anticancer agent and opioid receptoragonist.

In a preferred embodiment of the invention the administration regimenand thus the period within a therapeutically effective plasma level ofthe anticancer agent is defined by the conventional therapy regimen.

In a further aspect of the invention the patient treated with thecombination according to the invention has received a pre-treatmentcomprising an anticancer agent.

In a more preferred embodiment the pre-treatment with the anticanceragent has been discontinued or even terminated.

In a further preferred embodiment the pre-treatment has been terminateddue to resistance against the anticancer treatment.

In a preferred embodiment of the invention the period with atherapeutically effective plasma level of the anticancer agent lasts forat least 1 day, preferably 3 days, and more preferably for at least 5days.

In one embodiment of the invention the period with a therapeuticallyeffective plasma level for the opioid receptor agonist is at least twoweeks, more preferably four weeks and even more preferably represents achronic treatment.

Within the context of the present invention the term “chronic treatment”is defined as a opioid receptor agonist treatment with an administrationperiod above four weeks, which preferably lasts over several months. Ina further embodiment this chronic treatment differs from theconventional therapy regimen as prescribed or known to the personskilled in art. or is published in therapeutic guidelines ofassociations or federations like Deutsche Krebshilfe or DeutscheKrebsgesellschaft; NCCN, NCI or similar health or cancer organizationsor the guidelines of producer or distributer of drugs which are used fortreatment of cancer

Within the context of the present invention the use of at least oneanticancer agent refers to the use of one or more anticancer agents tobe given in combination with the opioid receptor agonist according theinvention. Thus, the combination includes the use of one, two, three,four, five or even more anticancer agents.

Generally, it is known, that apoptosis can be induced via two mainbiochemical pathways. The “death receptor pathway” (or extrinsicpathway) includes the TNF-receptor-induced (tumour necrosis factor)model and the Fas-receptor-induced model (the Fas-receptor is also knownas Apo-1 or CD95). Bindings to these receptors result in the formationof death-inducing signalling pathways in the cell, including theactivation of caspase-8. The “mitochondrial pathway” (or intrinsicpathway) involves the release of cytochrome c from mitochondria, bindingof Apaf-1 and activation of procaspase-9. Several regulators are knownto activate or deactivate the apoptosis pathways, such as thepro-apoptotic proteins Bax and Bak or the anti-apoptotic proteins Bcl-2,Bcl-_(XL) or XIAP.

In one embodiment of the invention the opioid receptor agonists induceapoptosis by cleavage of caspase-3 and poly(ADP-ribose) polymerase(PARP) in the tumour cell, and/or cleavage of caspase-9 and downregulation of X-linked inhibitor of apoptosis protein (XIAP), and/ordown regulation of the B-cell lymphoma-extra large protein (Bcl-_(XL)).

According to a preferred embodiment of the invention, the opioidreceptor agonist is a member of the methadone group, comprisingDA-methadone, levomethadone, levacetylmethadol and piritramide.

In the context of the present invention the term “methadone group”relates to opioids which are derivatives of 3,3-diphenylpropylamine.These compounds possess the 3,3-diphenylamine core structure as shown bythe following formula (I):

wherein R₁ is an aliphatic ketone, a 3-acetoxypropyl residue, a cyanogroup, or a 1-pyrrolidino-methylketone, —(C═O)C₂H₅, R₂ and R₃ are CH₃ ortogether forming a heterocyle, preferably a morpholino group, and R₄ isH or an alkyl residue, being preferably CH₃.

A non-limited list of examples for compounds of the methadone groupincludes methadone, normethadone, dextromoramide, isomethadone,acetylmethadol, alphacetylmethadol, levoacetylmethadol, premethadone,racemoramid, phenadoxone, dextropropoxyphene, dipipanone, benzitramide,piritramide, loperamide, themalon (which represents a3,3-dithiophenylpropylamine) and levomoramid.

All these opioids can be used as salts. The racemic form of DA-methadoneis preferably provided in form of a hydrochloride. In a preferredembodiment of the invention, the opioid methadone induces apoptosis incancer cells via the mitochondrial pathway.

In one embodiment of the invention the opioid receptor agonist isselected from the list consisting of compounds of the methadone groupsuch as D/L-methadone, D-methadone, L-methadone, normethadone, fentanylderivatives such as fentanyl, sufentanyl, lofenantil, alfentanil,remifentanil, ohmefentanyl and carfentanyl; morphinane compounds such asmorphine, codeine, heroine, dextrallorphane, dextromethorphan,dextrophanol, dimemorfan, levalorphanol, butorphanol,levofurethylnormorphanol, levomethorphane, levophenacylmorphane,levorphanol, methorphane, morphanol, oxilorphan, phenomorphan, andxorphanol, benzomorphane derivatives such as 5,9-DEHB, alazocine,anazocine, bremazocine, butinazocine, carbazocine, cogazocine,cyclazocine, dezocine, eptazocine, etazocine, ethylketocyclazocine,fluorophen, gemazocine, ibazocine, ketazocine, ketocyclazozine,metazocine, moxazocine, pentazocine, phenazocine, quadazocine,thiazocine, tonazocine, volazocine and 8-CAC; endogenous opioids such asendorphins (which can be alpha-, beta-, gamma- or delta-endorphins),enkephalins such as Met-enkephalin, Leu-enkephalin and methorphamid,dynorphins such as dynorphin A, dynorphin B or alpha-neoendorphin,nociceptin, dermorphins, morphiceptin, beta-caomorphine-5, DALAMID,DADLE, DADL DSLT, DSLET, DTLET, DAGO, DAMGO, DALCE, DAMME, DALDA, DPDPE,FK 33-824, [D-Met2,Pro5]enkephalin-amide, biphalin, and endomorphinessuch as endomoprhin-1 and endomoprhin-2; furthermore all fragmentsderived from the protein proopiomelanocortin (POMC) such asbeta-lipotropin, beta-LPH-[61-64], beta-LPH-[61-65]-NH₂,(Met(O)65)-beta-LPH-[61-65], beta-LPH-[61-69], and beta-LPH-[61-69];4-phenylpiperidine derivatives such as pethidine, ketobemidone,anileridine, piminodine, phenoperidine, furethidine, alpha-prodin,trimeperidine, including 4-phenylpyrrolidine derivatives such asprofadol and 4-phenylazepanderivates such as meptazinol; cyclohexanederivatives such as tilidine, U-50488, tramadol and tapentadol.

In a preferred embodiment of the invention the opioid receptor agonistsof the invention are capable of inhibiting cell proliferation.

In a further embodiment of the invention the opioid receptor agonist iscombined with at least one additional opioid receptor agonist. As aresult the combination of the invention consists of two, three, four ormore opioid receptor agonists. Preferably a combination of two differentopioid receptor agonists is used. It could be demonstrated that thecombined use of different opioids leads to a synergistic pro-apoptoticeffect on cancer cells (see Example 38 and FIG. 56).

In a preferred embodiment said combination of opioid receptor agonistscomprises morphine and fentanyl. Preferably said combination consists ofmorphine and fentanyl. The synergistic effect of morphine and fentanylwas e.g. shown for the leukemia cell line HL60 (see Example 38 and FIG.56).

In a preferred embodiment the methadone, preferably the D,L-methadoneand most preferably the hydrochloride form of D,L-methadone is given tothe patient in particular to yield a plasma level which is between 0.05μg/mL and 3 μg/mL.

In a further preferred embodiment the methadone, preferably theD,L-methadone and most preferably the hydrochloride form ofD,L-methadone is given to the patient in particular to yield a plasmalevel which is between 0.01 μg/mL and 3 μg/mL.

In one embodiment of the invention the anticancer agent is selected fromthe list consisting of intercalating substances such as anthracyclinedoxorubicin, idarubicin, epirubicin, and daunorubicin; topoisomeraseinhibitors such as irinotecan, topotecan, camptothecin, lamellarin D,etoposide, teniposide, mitoxantrone, amsacrine, ellipticines andaurintricarboxylic acid; nitrosourea compounds such as carmustine(BCNU), lomustine (CCNU), streptozocin; nitrogen mustards such ascyclophosphamide, mechlorethamine, uramustine, bendamustine, melphalan,chlorambucil, mafosfamide, trofosfamid and ifosfamide; alkyl sulfonatessuch as busulfan and treosulfan; alkylating agents such as procarbazin,dacarbazin, temozolomid and thiotepa; platinum analogues such ascisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, andtriplatin tetranitrate; microtubule disruptive drugs such asvinblastine, colcemid and nocodazole; antifolates like methotrexate,aminopterin, dichloromethotrexat, pemetrexed, raltitrexed andpralatrexate; purine analogues like azathioprine, mercaptopurine,thioguanine, fludarabine, fludarabine phosphate, pentostatin andcladribine; pyrimidine analogues like 5-fluorouracil, floxuridine,cytarabine, 6-azauracil, gemcitabine, capecitabine; taxane and taxaneanalogues like paclitaxel and docetaxel; steroid hormones likegestagene, androgene, glucocorticoids, dexamethasone, prednisolone, andprednisone; anti-cancer peptides including radioactively labeledpaptides and peptide-drug conjugates; anti-cancer antibodies includingradioactively-labelled antibodies and antibody-drug conjugates such asbevacizumab, cetuximab, panitumumab, rituximab, ipilimumab, alemtuzumab,ofatumumab, gemtuzumab-ozogamicin, brentuximab vedotin, ⁹⁰Y-ibritumomabtiuxetan, ¹³¹1-tositumomab, or trastuzumab, alpha, beta or gammairradiation; including particle radiation.

The above listed anticancer agents comprise also modifications such asPEGylation and formulations such as the use of liposomes (i.e. PEGylatedliposomal doxorubicin).

In a further embodiment the anticancer agent can be a radioactivelylabeled chemical compound, peptide, protein or monoclonal antibody,wherein the radioactive label could emit alpha, beta or gamma rays andalso ionizing particles.

In a preferred embodiment of the invention the anticancer agent ismethotrexate, cytarabine, gemcitabine, paclitaxel, docetaxel,carboplatin, oxaliplatin, etoposide, vincristine, fludarabine especiallycisplatin, doxorubicin, anthracycline, idarubicin, daunorubicin,epirubicin, or alpha-, beta-, or gamma irradiation.

When treating a cancer entity for which an induction of the opioidreceptor is desired the patient is preferably treated with an anticanceragent selected from the group consisting doxorubicin, idarubicin,epirubicin, daunorubicin, carboplatin, oxaliplatin, cisplatin,etoposide, methotrexate, cytarabine, teniposide, gemcitabine,paclitaxel, rituximab and trastuzumab.

In one embodiment of the invention the patient who is treated with thecombination of the invention suffers from a neoplasm as classifiedaccording the International statistical classification of Diseases andrelated health problems 10^(th) Revision (ICD-10), wherein the neoplasmis from the group consisting of malignant neoplasms of classes COO toC97, in situ neoplasms of classes DOO to D09, benign neoplasms ofclasses D10 to D36, and neoplasms of uncertain or unknown behaviour ofclasses D37 to D48.

The classes are defined as follows: (C00) Malignant neoplasm of lip,(C01) Malignant neoplasm of base of tongue, (C02) Malignant neoplasm ofother and unspecified parts of tongue, (C03) Malignant neoplasm of gum,(C04) Malignant neoplasm of floor of mouth, (C05) Malignant neoplasm ofpalate, (C06) Malignant neoplasm of other and unspecified parts ofmouth, (C07) Malignant neoplasm of parotid gland, (C08) Malignantneoplasm of other and unspecified major salivary glands, (C09) Malignantneoplasm of tonsil, (C10) Malignant neoplasm of oropharynx, (C11)Malignant neoplasm of nasopharynx, (C12) Malignant neoplasm of piriformsinus, (C13) Malignant neoplasm of hypopharynx, (C14) Malignant neoplasmof other and ill-defined sites in the lip, oral cavity and pharynx,(C15) Malignant neoplasm of esophagus, (C16) Malignant neoplasm ofstomach, (C17) Malignant neoplasms of small intestine, (C18) Malignantneoplasm of colon, (C19) Malignant neoplasm of rectosigmoid junction,(C20) Malignant neoplasm of rectum, (C21) Malignant neoplasms of anusand anal canal, (C22) Malignant neoplasms of liver and intrahepatic bileducts, (C23) Malignant neoplasm of gallbladder, (C24) Malignant neoplasmof other and unspecified parts of biliary tract, (C25) Malignantneoplasm of pancreas, (C26) Malignant neoplasms of other and ill-definedDigestive Organs, (C30) Malignant neoplasm of nasal cavity and middleear, (C31) Malignant neoplasm of accessory sinuses, (C32) Malignantneoplasm of larynx, (C33) Malignant neoplasm of trachea, (C34) Malignantneoplasm of bronchus and lung, (C37) Malignant neoplasm of thymus, (C38)Malignant neoplasm of heart, mediastinum and pleura, (C39) Malignantneoplasms of other and ill-defined sites in respiratory system andintrathoracic organs, (C40-C41) Malignant neoplasms, bone and articularcartilage, (C43) Malignant melanoma of Skin, (C44) Other malignantneoplasms of skin, (C45) Mesothelioma, (C46) Kaposi's Sarcoma, (C47)Malignant neoplasm of peripheral nerves and autonomic nervous system,(C48) Malignant neoplasm of retroperitoneum and peritoneum, (C49)Malignant neoplasm of other connective and soft tissue, (C50) Malignantneoplasm of breast, (C51) Malignant neoplasm of vulva, (C52) Malignantneoplasm of vagina, (C53) Malignant neoplasm of cervix uteri, (C54)Malignant neoplasm of corpus uteri, (C55) Malignant neoplasm of uterus,part unspecified, (C56) Malignant neoplasm of ovary, (C57) Malignantneoplasms of other and unspecified female and genital organs, (C58)Malignant neoplasm of placenta, (C60) Malignant neoplasm of penis, (C61)Malignant neoplasm of prostate, (C62) Malignant neoplasm of testis,(C63) Malignant neoplasm of other and unspecified male genital organs,(C64) Malignant neoplasm of kidney, except renal pelvis, (C65) Malignantneoplasm of renal pelvis, C66) Malignant neoplasm of ureter, (C67)Malignant neoplasm of bladder, (C68) Malignant neoplasm of other andunspecified urinary organs, (C69) Malignant neoplasms of eye and adnexa,(C70) Malignant neoplasm of meninges, (C71) Malignant neoplasm of brain,(C72) Malignant neoplasm of spinal cord, cranial nerves and other partsof central nervous system, (C73) Malignant neoplasm of thyroid gland,(C74) Malignant neoplasm of adrenal gland, (C75) Malignant neoplasm ofother endocrine glands and related structures, (C76) Malignant neoplasmof other and ill-defined sites, (C77) Secondary and unspecifiedmalignant neoplasm of lymph nodes, (C78) Secondary malignant neoplasm ofrespiratory and digestive organs, (C79) Secondary malignant neoplasm ofother sites, (C80) Malignant neoplasm without specification of site,(C81) Hodgkin's Disease, (C82) Follicular non-Hodgkin's lymphoma(nodular), (C83) Diffuse non-Hodgkin's lymphoma, (C84) Peripheral andcutaneous T-cell lymphomas, (C85) Other and unspecified types ofnon-Hodgkin's lymphoma, (C88) Malignant immunoproliferative diseases,(C90) Multiple myeloma and malignant plasma cell neoplasms, (C91)Lymphoid leukemia, (C92) Myeloid leukemia, (C93) Monocytic leukemia,(C94) Other leukemias of specified cell type, (C95) Leukemia ofunspecified cell type, (C96) Other and unspecified malignant neoplasmsof lymphoid, haematopoietic and related tissue, (C97) Malignantneoplasms of independent (primary) multiple sites, (D00) Carcinoma insitu of oral cavity, oesophagus and stomach, (D01) Carcinoma in situ ofother and unspecified digestive organs, (D02) Carcinoma in situ ofmiddle ear and respiratory system, (D03) Melanoma in situ, (D04)Carcinoma in situ of skin, (D05) Carcinoma in situ of breast, (D06)Carcinoma in situ of cervix uteri, (D07) Carcinoma in situ of other andunspecified genital organs, (D09) Carcinoma in situ of other andunspecified sites, (D10) Benign neoplasm of mouth and pharynx, (D11)Benign neoplasm of major salivary glands, (D12) Benign neoplasm ofcolon, rectum, anus and anal canal, (D13) Benign neoplasm of other andill-defined parts of digestive system, (D14) Benign neoplasm of middleear and respiratory system, (D15) Benign neoplasm of other andunspecified intrathoracic organs, (D16) Benign neoplasm of bone andarticular cartilage, (D17) Benign lipomatous neoplasm, (D18) Haemangiomaand lymphangioma, any site, (D19) Benign neoplasm of mesothelial tissue,(D20) Benign neoplasm of soft tissue of retroperitoneum and peritoneum,(D21) Other benign neoplasms of connective and other soft tissue, (D22)Melanocytic naevi, (D23) Other benign neoplasms of skin, (D24) Benignneoplasm of breast, (D25) Leiomyoma of uterus, (D26) Other benignneoplasms of uterus, (D27) Benign neoplasm of ovary, (D28) Benignneoplasm of other and unspecified female genital organs, (D29) Benignneoplasm of male genital organs, (D30) Benign neoplasm of urinaryorgans, (D31) Benign neoplasm of eye and adnexa, (D32) Benign neoplasmof meninges, (D33) Benign neoplasm of brain and other parts of centralnervous system, (D34) Benign neoplasm of thyroid gland, (D35) Benignneoplasm of other and unspecified endocrine glands, (D36) Benignneoplasm of other and unspecified sites, (D37) Neoplasm of uncertain orunknown behaviour of oral cavity and digestive organs, (D38) Neoplasm ofuncertain or unknown behaviour of middle ear and respiratory andintrathoracic organs, (D39) Neoplasm of uncertain or unknown behaviourof female genital organs, (D40) Neoplasm of uncertain or unknownbehaviour of male genital organs, (D41) Neoplasm of uncertain or unknownbehaviour of urinary organs, (D42) Neoplasm of uncertain or unknownbehaviour of meninges, (D43) Neoplasm of uncertain or unknown behaviourof brain and central nervous system, (D44) Neoplasm of uncertain orunknown behaviour of endocrine glands, (D45) Polycythaemia vera, (D46)myelodysplastic syndromes, (D47) Other neoplasms of uncertain or unknownbehaviour of lymphoid, haematopoietic and related tissue, (D48) Neoplasmof uncertain or unknown behaviour of other and unspecified sites.

In a specific embodiment of the invention the patient who is treatedwith the combination of the invention suffers from metastases.

In a preferred embodiment of the invention the patient who is treatedwith the combination of the invention suffers from a neoplasm selectedfrom list of classes consisting of C25, C50, C56, C71, C91, and C92.

In a more preferred embodiment the patient suffers from a neoplasmselected from the list comprising of acute lymphoblastic leukemia(C91.0), B-cell chronic lymphatic leukemia (C91.2), acute promyelocyticleukemia (C92.4), acute myeloid leukemia (C92.0) chronic myeloidleukemia (C92.1), all forms of glioblastoma (C71), all forms ofpancreatic cancer (C25), all forms of ovarian cancer (C56), classes ofbreast cancer (C50) and tumour stem cells such as glioblastomainitiating stem cells.

In a further embodiment of the invention the patient suffers from abreast cancer resistant to HER2-targeted therapies, like e.g. aTrastuzumab resistant breast cancer.

In a preferred embodiment the patient suffering from acute lymphoblasticleukemia (C91.0) is treated with the combination according the inventionincluding an anticancer agent selected from the list consisting ofmethotrexate, cytarabine, carboplatin, oxaliplatin, vincristine,fludarabine, being preferably cisplatin, anthracycline doxorubicin,idarubicin, daunorubicin, epirubicin, etoposide, gemcitabine,paclitaxel, docetaxel or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering from acutelymphoblastic leukemia (C91.0) is treated with the combination accordingthe invention including an opioid receptor agonist selected from thelist consisting of D,L-methadone, buprenorphine, fentanyl, and morphine,being preferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from acute lymphoblastic leukemia (C91.0) is treated with thecombination comprising D,L-methadone and etoposide or D,L-methadone anddoxorubicin.

In a preferred embodiment the patient suffering from B-cell chroniclymphatic leukemia (C 91.2) is treated with the combination accordingthe invention including an anticancer agent selected from the listconsisting of methotrexate, cytarabine, carboplatin, oxaliplatin,vincristine, fludarabine, being preferably cisplatin, anthracyclinedoxorubicin, idarubicin, daunorubicin, epirubicin, etoposide,gemcitabine, paclitaxel, docetaxel or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering from B-cellchronic lymphatic leukemia (C 91.2) is treated with the combinationaccording the invention including an opioid receptor agonist selectedfrom the list consisting of D,L-methadone, buprenorphine, fentanyl, andmorphine, being preferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from B-cell chronic lymphatic leukemia (C 91.2) is treatedwith the combination comprising D,L-methadone and fludarabine, orbuprenorphine and fludarabine or fentanyl and fludarabine or morphineand fludarabine.

In a preferred embodiment the patient suffering from acute promyelocyticleukemia (C92.4) is treated with the combination according the inventionincluding an anticancer agent selected from the list consisting ofmethotrexate, cytarabine, carboplatin, oxaliplatin, vincristine,fludarabine, being preferably cisplatin, anthracycline, doxorubicin,idarubicin, daunorubicin, epirubicin, etoposide, gemcitabine,paclitaxel, docetaxel or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering from acutepromyelocytic leukemia (C92.4) is treated with the combination accordingthe invention including an opioid receptor agonist selected from thelist consisting of D,L-methadone, buprenorphine, fentanyl, and morphine,being preferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from acute promyelocytic leukemia (C92.4) is treated with thecombination comprising D,L-methadone and doxorubicin or buprenorphineand doxorubicin or fentanyl and doxorubicin or morphine and doxorubicin.

In a preferred embodiment the patient suffering from acute myeloidleukemia (C92.0) is treated with the combination according the inventionincluding an anticancer agent selected from the list consisting ofmethotrexate, cytarabine, carboplatin, oxaliplatin, vincristine,fludarabine, being preferably cisplatin, anthracycline, doxorubicin,idarubicin, daunorubicin, epirubicin, etoposide gemcitabine, paclitaxel,docetaxel or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering from acutemyeloid leukemia (C92.0) is treated with the combination according theinvention including an opioid receptor agonist selected from the listconsisting of D,L-methadone, buprenorphine, fentanyl, and morphine,being preferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from acute myeloid leukemia (C92.0) is treated with thecombination comprising D,L-methadone and doxorubicin or buprenorphineand doxorubicin or fentanyl and doxorubicin or morphine and doxorubicin.

In a preferred embodiment the patient suffering from chronic myeloidleukemia (C92.1) is treated with the combination according the inventionincluding an anticancer agent selected from the list consisting ofmethotrexate, cytarabine, carboplatin, oxaliplatin, vincristine,fludarabine, being preferably cisplatin, anthracycline doxorubicin,idarubicin, daunorubicin, epirubicin, etoposide, gemcitabine,paclitaxel, docetaxel or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering from chronicmyeloid leukemia (C92.1) is treated with the combination according theinvention including an opioid receptor agonist selected from the listconsisting of D,L-methadone, buprenorphine, fentanyl, and morphine,being preferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from chronic myeloid leukemia (C92.1) is treated with thecombination comprising D,L-methadone and imatinib or buprenorphine andimatinib or fentanyl and imatinib or morphine and imatinib.

In another preferred embodiment of the invention the patient sufferingfrom chronic myeloid leukemia (C92.1) is treated with the combinationcomprising D,L-methadone and fludarabine or buprenorphine andfludarabine or fentanyl and fludarabine or morphine and fludarabine.

In a further embodiment of the invention the patient suffering fromleukemia is treated with at least one further opioid receptor agonist inaddition to the combination of the invention.

Hence, the patient is treated with at least two opioid receptoragonists. This strategy is based on the finding that the combination ofdifferent opioids shows a synergistic pro-apoptotic effect on cancercell lines (see FIG. 56).

In a preferred embodiment the combination of the invention comprisesmorphine, fentanyl and at least one anticancer agent and in a furtherembodiment the combination consists of morphine, fentanyl and a furtheranticancer agent. The synergistic effect of morphine and fentanyl wase.g. shown for the leukemia cell line HL60 (see Example 38).

In a preferred embodiment the patient suffering from glioblastoma (C71)is treated with the combination according the invention including ananticancer agent selected from the list consisting of methotrexate,cytarabine, carboplatin, oxaliplatin, vincristine, fludarabine, beingpreferably cisplatin, temozolomide, anthracycline, doxorubicin,idarubicin, daunorubicin, epirubicin, etoposide, gemcitabine,paclitaxel, docetaxel, or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering fromglioblastoma (C71) is treated with the combination according theinvention including an opioid receptor agonist selected from the listconsisting of D,L-methadone, buprenorphine, fentanyl, and morphine,being preferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from glioblastoma (C71) is treated with the combinationcomprising D,L-methadone and doxorubicin.

In a preferred embodiment said doxorubicin is given in a formulationthat enhances the transfer of the doxorubicin across the blood brainbarrier, For the skilled person there are several formulation strategiesavailable to enable or enhance BBB transfer. As an example, thedoxorubicin could be packed into liposomes or bound to transferrin.

In another preferred embodiment of the invention the patient sufferingfrom glioblastoma (C71) is treated with the combination comprisingD,L-methadone and daunorubicin, with a combination comprisingD,L-methadone and idarubicin, or with a combination comprisingD,L-methadone and temozolomide.

In a preferred embodiment the patient suffering from glioblastomainitiating stem cells are treated with the combination according theinvention including an anticancer agent selected from the listconsisting of methotrexate, cytarabine, carboplatin, oxaliplatin,vincristine, fludarabine, being preferably cisplatin, anthracycline,doxorubicin, idarubicin, daunorubicin, epirubicin, etoposide,gemcitabine, paclitaxel, docetaxel or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering fromglioblastoma initiating stem cells are treated with the combinationaccording the invention including an opioid receptor agonist selectedfrom the list consisting of D,L-methadone, buprenorphine, fentanyl, andmorphine, being preferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from glioblastoma initiating stem cells are treated with thecombination comprising D,L-methadone and doxorubicin.

In a preferred embodiment the patient suffering from pancreatic cancer(C25) is treated with the combination according the invention includingan anticancer agent selected from the list consisting of methotrexate,cytarabine, carboplatin, oxaliplatin, vincristine, fludarabine, beingpreferably cisplatin, oxaliplatin, anthracycline doxorubicin,idarubicin, daunorubicin, epirubicin, etoposide, gemcitabine,paclitaxel, docetaxel or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering from pancreaticcancer (C25) is treated with the combination according the inventionincluding an opioid receptor agonist selected from the list consistingof D,L-methadone, buprenorphine, fentanyl, and morphine, beingpreferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from pancreatic cancer (C25) is treated with the combinationcomprising D,L-methadone and cisplatin.

In a further preferred embodiment of the invention the patient sufferingfrom pancreatic cancer (C25) is treated with the combination comprisingD,L-methadone and oxaliplatin.

In one embodiment of the invention the patient suffering from cancer istreated with a combination comprising D,L-methadone and temozolomide.

In a preferred embodiment the patient suffering from ovarian cancer(C56) is treated with the combination according the invention includingan anticancer agent selected from the list consisting of methotrexate,cytarabine, carboplatin, oxaliplatin, vincristine, fludarabine, beingpreferably cisplatin, carboplatin, anthracycline doxorubicin,idarubicin, daunorubicin, epirubicin, etoposide, gemcitabine,paclitaxel, docetaxel or alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering from ovariancancer (C56) is treated with the combination according the inventionincluding an opioid receptor agonist selected from the list consistingof D,L-methadone, buprenorphine, fentanyl, and morphine, beingpreferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from ovarian cancer (C56) is treated with the combinationcomprising D,L-methadone and cisplatin.

In another embodiment, the patient who is treated with the combinationof D,L-methadone and cisplatin suffers from a cisplatin resistantovarian cancer.

In a preferred embodiment the patient suffering from breast cancer (C50)is treated with the combination according the invention including ananticancer agent selected from the list consisting of methotrexate,cytarabine, carboplatin, oxaliplatin, vincristine, fludarabine, beingpreferably cisplatin, anthracycline doxorubicin, idarubicin,daunorubicin, epirubicin, etoposide, gemcitabine, paclitaxel, docetaxelor alpha, beta or gamma irradiation.

In a further preferred embodiment the patient suffering from breastcancer (C50) is treated with the combination according the inventionincluding an opioid receptor agonist selected from the list consistingof D,L-methadone, buprenorphine, fentanyl, and morphine, beingpreferably D,L-methadone.

In an even more preferred embodiment of the invention the patientsuffering from breast cancer (C50) is treated with the combinationcomprising D,L-methadone and cisplatin.

In a further preferred embodiment of the invention the patient sufferingfrom breast cancer (C50), which preferably is a breast cancer resistantto HER2 targeted therapies, such as

Trastuzumab resistant breast cancer, is treated with the combinationcomprising D,L-methadone and doxorubicin.

In another embodiment of the invention the patient suffering fromprostate cancer (C62) is treated with the combination comprisingD,L-methadone and cisplatin.

In one embodiment of the invention the patient suffering from leukemiais treated with a combination of D,L-methadone and one of the followinganticancer agents: etoposide, cytarabine, methotrexate,cyclophosphamide, thioguanine, gemcitabine, paclitaxel, docetaxel orvincristine.

In another embodiment the cancer to be treated is a neoplasm accordingthe International classification of Diseases for Oncology ICD-O in theactual version ICD-O-3 from 2000. Alternatively, the cancer to betreated is a cancer as included in the TNM Classification of MalignantTumours (TNM), which represents a cancer staging system that describesthe extent of cancer in a patient's body. In a further alternative, thecancer to be treated is disclosed by Boecker et al., 2008 in chapter 6(Pathologie, Elsevier, Urban & Fischer, p. 167-218), which isincorporated by reference in its entirety.

In a preferred embodiment of the invention the patient that is treatedwith said combination suffers from non-solid tumours from the groupconsisting of leukemia, breast cancer, skin cancer, bone cancer,prostate cancer, liver cancer, lung cancer, brain cancer, cancer of thelarynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal,neural tissue, head and neck, colon, stomach, bronchi, kidneys, basalcell carcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteosarcoma, Ewing's sarcoma,veticulum cell sarcoma, myeloma, giant cell tumour, small-cell lungtumour, islet cell tumour, primary brain tumour, acute and chroniclymphocytic and granulocytic tumours, hairy-cell tumour, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas,intestinal ganglioneuromas, hyperplastic corneal nerve tumour, marfanoidhabitus tumour, Wilms' tumour, seminoma, ovarian tumour, leiomyomata,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumour, polycythermia vera,adenocarcinoma, glioblastoma multiforme, leukemia, lymphomas, malignantmelanomas, and epidermoid carcinomas.

In a further preferred embodiment of the invention the patient to betreated suffers from a neoplasm selected from the group consisting ofpancreatic carcinoma, hepatoblastoma, colon carcinoma, (small cell lungcancer, melanoma, mamma carcinoma, ovarian carcinoma, prostatecarcinoma, glioblastoma, acute lymphoblastic leukemia, acute myeloidleukemia, chronic myeloid leukemia, chronic lymphocytic leukemia,pro-forms of leukemia, hairy cell leukemia, Hodgkin's disease,Non-Hodgkin lymphoma, lymphoma, tumour stem cells,glioblastoma-initiating stem cells and multiple myeloma.

In another embodiment of the invention the patient exhibits either anintrinsic or an acquired resistance.

Accordingly, in the context of the present invention a “resistance” caneither be total or partly; in other words, the patients consideredtreatable according to the invention can exhibit a reduced sensitivityor even a full lack of sensitivity to conventional anticancertreatments. These patients can also be determined as “non-responders” or“poor-responders”.

A further synonym for a “resistant” cancer or tumour is a “refractory”type of cancer, which can also be either completely or partlyrefractory. Intrinsic resistance can thus also be determined as a“primary refractory cancer”. A particular form of refractory orresistant cancer cells are the so called “kinetically refractory cells”;a phenomenon known e.g. from leukemia cells, when the cells are at firstkilled, but reproduce fast that an effective treatment is hardlypossible.

As used in the context of the present invention the term “conventional”treatment or therapy refers to the currently accepted and widely usedtherapeutic treatment of a certain type of cancer, based on the resultsof past researches and/or regulatory approval.

Conventional anticancer drugs include cytotoxic and cytostatic agents,which kill the cancer cells or reduce and/or stop their growth orproliferation. The modes of action of these anticancer drugs can vary;examples are antimetabolites (e.g. cytarabine, methotrexate,mercaptopurine or clofarabine), DNA cross-linking agents (e.g. cisplatinand its derivatives), DNA intercalating substances (e.g. doxorubicin),Topoisomerase poisons (e.g. etoposide), kinase inhibitors (e.g.cetuximab), steroids (e.g. dexamethasone) or mitotic inhibitors (e.g.vincristine). One example for a conventional anticancer treatment ofleukemia is the administration of doxorubicin or rituximab.

The conventional radiotherapy can also include radiation therapy, whichmeans the use of high-energy radiation from x-rays, alpha, beta andgamma rays, Auger electrons, Ultraviolet rays, neutrons, protons, andother sources to kill cancer cells and shrink tumours. Radiation mayoriginate from an outside the body device (external-beam radiationtherapy), or it may originate from radioactive sources placed in thebody in the vicinity of the cancer cells (internal radiation therapy).Systemic radiation therapy uses a radioactive substance, such as aradiolabeled monoclonal antibody, that travels in the blood stream tothe target tissue. Radio resistant cancer cells do not or only partlyrespond to these treatments.

As outlined in detail above, according to one embodiment of theinvention the opioid receptor agonists are applied for overcoming or“breaking” the intrinsic or acquired resistance of cancer cells toconventional anticancer treatments and/or radiation treatment orapoptosis resistance. In one embodiment of the invention cancer cellsconsidered treatable according to the invention express an opioidreceptor, in particular the p opioid receptor.

According to the invention, the terms “resistance”, “radioresistance” or“chemoresistance” are defined as a reduced sensitivity of a cancer cellto at least one conventional cancer therapy, i.e. either an anticancerdrug or radiotherapy. A patient suffering from such a cancer isdetermined as a “resistant” cancer patient. Since the resistance can beintrinsic or acquired the observed reduction in sensitivity is eithercompared to fully sensitive “normal” cancer cells, which are responsiveto the therapeutically effective dosage of the applied anticancer drugand/or radiation compared to the original sensitivity upon therapyonset. In the later case the resistance manifests either in a diminishedamount of tumour regression for the same dose (either of the radiationor the anticancer drug) or an increased dose which is necessary for anequal amount of tumour regression.

In another embodiment of the invention the patient exhibits one or moreof the subsequent resistances: apoptosis resistance, multi-drugresistance, anticancer drug resistance, cytotoxic drug resistance,resistance to reactive oxygen species, resistance to DNA damagingagents, resistance to toxic antibodies, doxorubicin resistance, singleor cross resistance, irradiation resistance (e.g. alpha, beta, gamma orAuger electrons).

In a particular embodiment the patient is resistant to one or more ofthe following drug substances: methotrexate, cytarabine, thioguaninecisplatin, oxaliplatin, etoposide, vincristine, paclitaxel, carboplatin,teniposide, dexamethasone, prednisolone, cyclophosphamide,diphosphamide, doxorubicin, epirubicin, daunorubicin, idarubicin,mercaptopurine, fludarabine, gemcitabine, temozolomide, anti-HER2, andanti-CD20.

In one embodiment of the invention the anticancer agent that isadministered together with the opioid receptor agonist is given at adose, which is equal than or lower than the recommended dose for therespective cancer. The recommended dose is given by a conventionalcancer therapy without the administration of an opioid receptor agonist.Preferably, the respective dose of the anticancer agent from theperspective of the skilled person represents a suboptimal or subtherapeutic dose, which have the advantage for the patient to have lessside effects. The main effect is that the uptake of the dose of theanticancer drug is increased in the cancer cells, while the plasmaconcentration is on the level of the conventional therapy. This has theeffect that non responder to conventional therapy could be treated.

In a preferred embodiment of the invention the anticancer agent that isadministered together with the opioid receptor agonist is given at adose, which is 2 times lower, preferably 3, 5, 10, or 30 times lower andeven more preferably 100 times lower than the recommended dose for thetreatment of cancer using the anticancer agent only.

In a further preferred embodiment of the invention the anticancer agentthat is administered together with the opioid receptor agonist is givenat a dose, which is equal than or lower than the recommended dose forthe respective cancer, wherein the period of effective plasma levels ofthe anticancer agent is completely within the period of effective plasmalevels of the opioid receptor agonist. The recommended dose is given bya conventional cancer therapy without the administration of an opioidreceptor agonist.

In a further preferred embodiment of the invention the opioid receptoragonist is D/L-methadone and the anticancer agents are methotrexate anddexamethasone.

In a further embodiment of the invention, the opioids or opioid receptoragonist can be used as a composite with at least one anticancer drug.

In the context of the present invention, the term “anti-Her2” denotes toany ligand that binds to and interacts with the gene product of theHer-2/Neu gene. This encompasses antibodies such as Trastuzumab(herceptin) or any organic compounds.

A “composite” within the context of the present invention relates to apharmaceutical preparation comprising a therapeutically effective amountof any of the opioid receptor agonist (component A) as defined accordingto the invention and at least one further anticancer substance(component B). This “composite” can constitute a single composition orat least two compositions, which can be administered to the patientseither concomitantly or subsequently. The above mentioned substances arepreferably combined with methadone, more preferably with thehydrochloride form of D/L-methadone.

The composite of the invention can be advantageous for the effectivetreatment of cancer cells, since it can exhibit synergistic effectscompared to the single compositions. In particular composite withmethadone as component A and one of the agents as component B as followsis preferred: methotrexate, cytarabine, cisplatin, carboplatin,oxaliplatin, etoposide, vincristine, doxorubicin, idarubicin,epirubicin, daunorubicin, fludarabine. gemcitabine, paclitaxel,docetaxel, temozolomide, anti-CD20, anti-HER2. Moreover, combinatorialtreatment also comprising irradiation treatments is possible.

A further preferred composite consists of methadone as component A andtemozolomide as component B.

In a preferred embodiment of the invention opioids are used to treateither resistant or sensitive non-solid cancers, i.e. all haematologicalmalignancies affecting blood, bone marrow and lymph nodes, includingacute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloidleukemia, chronic lymphocytic leukemia and all pro-forms of leukemia,hairy cell leukemia, Hodgkin's disease, Non-Hodgkin lymphoma, lymphomaand multiple myeloma.

In a further aspect the invention provides a method for the selection ofa combination of an opioid receptor agonist and one or more anticancerdrugs. This method comprises the following steps:

-   -   (a) providing an vitro culture of cancer cells, cell lines or        primary cells, preferably isolated from a cancer biopsy or from        a liquid sample (such as e.g. blood, amniotic fluid, pleural        fluid, peritoneal fluid, or cerebrospinal fluid);    -   (b) optionally testing the cells from step (a) for expression of        opioid receptors;    -   (c) treating the cells from step (a) with an opioid agonist, or        at least one anticancer drug or a combination thereof;    -   (d) analysing the cells for cell death and/or expression of        opioid receptors (e) selecting the opioid receptor/anticancer        drug combination and preferably a dose for said combination        based on the desired extent of cell death/viability or        inhibiting proliferation; and/or    -   (f) selecting the anticancer agent and preferably a dose for        said anticancer agent which shows the desired extent of        induction of opioid receptors.

The in vitro cultured cancer cells can be an immortalized cell line,xenografted cells, a secondary or a primary cancer cell line or primarycells. In a preferred embodiment the cell line and/or cells is derivedfrom a cancer biopsy, in more preferred embodiment the biopsy or bloodsampling or cerebrospinal fluid sampling or pleural fluid sampling oramniotic fluid sampling or peritoneal fluid sampling is taken from thepatient to be treated with the combination according the invention. Thecancer cell line can represent a homogenous cell line based only on onecancer cell type or a heterogeneous cancer cell line comprising ofdifferent cell types.

The analysis of the opioid receptor expression in step (b) can beperformed by techniques which are known to the person skilled in art. Anon-limiting list of examples include immunofluorescence using anantibody or antibody fragment directed against said opioid receptor, theimmunoprecipitation of the opioid receptors, or the use of labelledopioid receptor ligands such as naloxone-fluorescein.

For the analysis of the cell viability and apoptosis in step (c) thereare several techniques which are known to the person skilled in art. Anon-limiting list of examples include (a) cytolysis or membrane leakageassays such as the lactate dehydrogenase assay, the propidium iodideassay, the Trypan blue assay, the 7-Aminoactinomycin D assay, (b)mitochondrial activity or caspase assays such as the Resazurin andFormazan (MTT/XTT) can assay for various stages in the apoptosis processthat foreshadow cell death, (c) functional assays which in the case ofred blood cells measure the cell deformability, osmotic fragility,haemolysis, ATP level, and haemoglobin content; (d) genomic andproteomic assays which include the analysis of the activation of stresspathways using DNA microarrays and protein chips.

In a further preferred embodiment the cell viability is measured by thepropidium iodide assay and the apoptosis is measured by determination ofhypodiploid DNA (subG1) and FSC/SSC analyses by flow cytometry.

In step (d) the cultured cells are preferably treated in parallelexperiments comprising the use of the opioid alone, the anticancer agentalone and a combination of the two substances. In a further embodimentthe potency of the effect is analysed by studying the dose dependency ofthe respective effect. In alternative experiments several anticanceragents can be combined to increase the anti-apoptotic effect or opioidreceptor expression or to reduce the side effect profile. In a furtherembodiment the initial selection of the test compounds will depend onthe characteristics of the tumour. Furthermore also the patientcharacteristics can be taken in consideration including the age, thesex, the body weight, co-morbidities, individual metabolic capabilities,allergies and incompatibilities, genetic predisposition, the course ofthe disease and the family history.

For the in vitro analysis the opioid receptor agonists as describedabove can be used for testing. Preferably, D,L-methadone, L-methadone,fentanyl, buprenorphine, morphine, codeine, oxycodone, tramadol andtapentadol are used.

In a preferred embodiment, an anti cancer agent is chosen which is wellknown to have an effect on the respective cancer cell type, cell line orcells.

When testing an anticancer agent alone, the cultured cells are analysedfor opioid receptor expression prior anticancer treatment and after theanticancer treatment under conditions which allow a comparison of theopioid receptor expression levels. Said comparison allows to identifyanticancer agents which increase the opioid receptor expression on therespective cancer cell.

The selection in step (e) prioritizes the drug combination and/or therespective doses in order to maximise the efficacy while retaining aside effect profile which is acceptable for the patient.

The selection in step (f) prioritizes an anticancer agent with regard toits ability to increase the opioid receptor expression on the cancercell. As a consequence the anti-apoptotic effect of the opioid agonist,as well as the anti-apoptotic affect of the anticancer agent ismaximised.

In the case that in step (c) the cell culture was treated with acombination of opioid receptor agonist and anticancer agent theprioritization of the combination which is used, is done under theaspect which combination of doses has the better lethal effect on thecells in culture. The combination with the highest lethal effect or ifobservable, the dose with a up to 10% less effect in lethality of cellscompared to the combination with the highest lethal effect on cells inculture but with the lower dose of opioid-receptor agonist should beused. FIG. 2 c for example shows that with doxorubicin in conventionaltherapeutical dose as described in the instruction leaflet aD,L-methadone dose of 0,1 μg/mL would be preferable.

In a further aspect the invention provides a method for selection of anopioid receptor agonist for the treatment of cancer comprising thefollowing steps:

-   -   (a) providing an vitro culture of cancer cells, cell lines or        primary cells, preferably isolated from a cancer biopsy or from        a liquid sample (such as e.g. blood, amniotic fluid, pleural        fluid, or peritoneal fluid or cerebrospinal fluid);    -   (b) optionally testing the cells from step (a) for expression of        opioid receptors;    -   (c) treating the cells from step (a);    -   (d) analysing the cells for cell death/viability or inhibition        of proliferation;    -   (e) selecting the opioid receptor/anticancer drug combination        and preferably a dose for said combination based on the desired        extent of cell death; and/or    -   (f) selecting the opioid receptor agonist and preferably a dose        for said opioid receptor agonist which shows the desired extent        of induction of cell death.

For this method the steps (a) to (d) can be performed by methods andstrategies as described above.

The analysis of the opioid receptor expression allows a selection ofcancer type which might be treated with an opioid receptor agonist. Dueto the in vitro treatment with an opioid receptor agonist, theindividual dose for the cancer in vivo treatment can be determined.

I. EXPERIMENTAL PROCEDURES

Drugs and Reagents For the in vitro experiments, D,L-methadonehydrochloride (D,L-methadone) and doxorubicin were purchased from Sigma(Taufkirchen, Germany), naloxone from Fagron GmbH&Co. KG (Barsbüttel,Germany), and pertussis toxin (PTX) from Calbiochem (Nottingham, UK).Prior to each experiment these substances were freshly dissolved insterile distilled water to ensure the constant quality of thepreparations. 3-lsobutyl-1-methylxanthine (IBMX, Sigma) was freshlydissolved in 0.01 N NaOH.

For in vivo application, we used D,L-methadone (Methaddict, Hexal,Germany) as 5 mg tablets purchased from the local pharmacy. The tabletswere pulverized and solubilized freshly before use in 10% Tween 80 insaline. Doxorubicin (Hexal) was purchased as injection solution (5mg/ml) and diluted freshly with saline to the appropriateconcentrations.

Cell Lines

The human B-cell leukemia (BCP-ALL) cell lines Tanoue, Reh and Nalm6were obtained from the DSMZ (Braunschweig, Germany) and cultured in RPMI1640 (Invitrogen) containing 10% heat inactivated FCS (Lonza, Verviers,Belgium), 1 mmol/L glutamine (Invitrogen), 1% penicillin/streptomycin(Invitrogen), 25 mmol/L HEPES (Biochrom) at 37° C., 95% air/5% CO₂. Inexperimental settings, the leukemia cells were seeded in a density of10,000 cells/mL.

Testing opioid receptor signaling Stimulation of opioid receptors (OR)by agonists like D,L-methadone leads to an activation of the inhibitoryG_(i)-protein. The α_(i)-subunit inactivates adenylyl cyclase (AC)resulting in a reduction of cAMP levels within the cell which in turnleads to apoptosis which might be mediated by several differentmodulators. Also the βγ-subunits of the G_(i)-protein modulate theactivity of different effectors like the inhibition of Ca²⁺- and theactivation of K⁺-channels. Naloxone as opioid receptor antagonistinhibits competitively opioid receptors. PTx (pertussis toxin)inactivates G_(i)-proteins and blocks downregulation of cAMP. IBMX(Isobutyl-1-methylxanthine) inhibits phosphodiesterase and increasescAMP levels.

Serum concentrations of methadone Determination of methadone in serumsamples was carried out after liquid/liquid extraction using a massspectrometer equipped with a gas chromatograph (GC/MS). As internalstandard d₉-methadone was added. The mass selective detector wasoperated in electron impact mode. Data were acquired in the selected-ionmonitoring mode. The analytes were identified with the following massesm/z 294, 223, 72 (target ion) for methadone and m/z 303, 226, and 78 ford₉-methadone with a limit of detection of 0,8 ng/ml and a limit ofquantification of 1,2 ng/ml.

Serum Concentrations of Doxorubicin

Determination of doxorubicin and its main metabolites in serum wereperformed as described previously (Hilger et al., 2005; Richly et al.,2006) Using this validated method, the quantification of doxorubicin,doxorubicinol, and 7-deoxy-doxorubicinolon was possible with a LLQ of0.2 ng/ml.

Patient-Derived-ALL Xenografts

For in vivo use ALL-SCID6 model was chosen. Fragments from in vivopassaged tumours were transplanted at day zero subcutaneously to 32 maleNOD/SCID/IL2ry null (NSG) mice. After randomization oral treatment (bygavage) with D,L-methadone was initiated one day later and performeddaily until the end of the experiment with increasing doses: 1^(st) week20 mg/kg/d, 2^(nd) week 30 mg/kg/d, 3^(rd) week 40 mg/kg/d, 4^(th) week60 mg/kg/d, 5^(th)-10^(th) week 2×60 mg/kg/inj.. The dose adaptation wasnecessary to avoid toxic deaths because of an overdosage ofD,L-methadone. The maximum tolerated dose of D,L-methadone in theemployed mouse strain is 2×60 mg/kg/inj. At day 46, 53, 60 and 76doxorubicin 3 mg/kg was administered i.v.. Tumour size was measuredtwice weekly at two dimensions and tumour volumes were calculatedaccording to the formula (length×width²)/2. Mean tumour volumes andstandard deviations were calculated per group. Treated to control values(T/C) in percent were calculated by relating mean tumour volumes of eachgroup at each measurement day to the controls. Individual body weightwas determined twice per week as parameter for tolerability and bodyweight changes in percent were calculated by relating the mean values ofeach group to the first measurement day.

Serum from D,L-methadone treated mice was taken 0.5, 1, 4 and 24 hoursafter last D,L-methadone treatment at day 76, respectively, and storedat −20° C. until the determination of methadone concentration. Mice weresacrificed at day 77 for ethical reasons.

For the in vitro investigations, cell suspensions of humanxenograft-derived-ALL-cells from patients with T-cell (ALL-SCID6,ALL-SCID3), B-cell (ALL-SCID7) and B-cell precursor (BCP,pre-B-ALL-SCID) acute leukemia were gained and cultivated in vitro andwere phenotypic and genotypic characterized as described (Borgmann etal., 2000). All animal experiments were approved by the localresponsible authorities (LaGeSo Berlin) and performed according to theguidelines for animal welfare in oncological experiments (Workman et al.2000).

Flow cytometric assay for determination of cell surface opioid-receptorsCells were washed in PBS supplemented with 1% FCS, centrifuged andresuspended in PBS/1% FCS containing naloxone-fluoresceine (0.05 mM,Invitrogen) (Hedin et al., 1997). After 30 min of incubation at RT, thecells were washed twice with PBS/1/0 FCS, centrifuged and resuspended inicecold PBS/1% FCS. Flowcytometry analysis was performed usingFACSCalibur (BD, Heidelberg, Germany).

Induction of Apoptosis

ALL cells were treated with D,L-methadone (≦3 μg/mL therapeutic plasmaconcentration) alone or in addition to doxorubicin in 175 cm² flasks or96-well plates. Further experiments were performed simultaneously afteraddition of 60 μg/mL naloxone, 200 μM IBMX or 200 ng/mL PTX. Afterdifferent points in time, apoptosis rates were measured by flowcytometry(Carbonari et al., 1994; Nicoletti et al., 1991). To determineapoptosis, cells were lysed with Nicoletti-buffer containing sodiumcitrate (0.1%), Triton X-100 (0.1%) and propidium iodide (50 μg/mL) asdescribed by Nicoletti (Nicoletti et al., 1991). Apoptotic cells weredetermined by hypodiploid DNA (subG1) or forward scatter/side scatteranalysis (Carbonari et al., 1994). The percentage of specific apoptosiswas calculated as follows: 100×[experimental dead cells (%)−spontaneousdead cells in medium (%)]/[100% -spontaneous dead cells in medium (%)].The spontaneous dead cells were in the rage of 5 to 10% using celllines. The viability of the untreated patient cells (spontaneous deadcells) was less than 35% at 24 h and 48h.

General Caspase Inhibition by zVAD.fmk

For inhibition of apoptosis, leukemia cells were treated with thepancaspase inhibitor of caspases, zVAD.fmk(benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone;Enzyme-Systems-Products, Dubli, USA) as described (Friesen et al.,2007). 50 μM zVAD.fmk was added to the cells 1h before stimulation withD,L-methadone and doxorubicin. After different time points, thepercentage of apoptotic cells was determined by FSC/SSC analysis viaflowcytometry(Carbonari et al., 1994).

Western Blot Analysis

Western blot analyses were performed as described (Classen et al., 2003;Friesen et al., 2004). Whole cell lysates were immunodetected for PARP,caspase-3, caspase-9, caspase-2, XIAP, Bcl-x_(L) and β-actin usingrabbit-anti-PARP-polyclonal-antibody (1:5000, Roche),mouse-anti-caspase-2-monoclonal-antibody (1:1000,BD-Transduction-Laboratories, Heidelberg, Germany),anti-XIAP-monoclonal-antibody (1:1000, BD-Transduction-Laboratories),mouse-anti-caspase-3-monoclonal-antibody (1:1000, Cell-Signaling,Boston, Mass./USA), rabbit-anti-caspase-9-polyclonal-antibody (1:1000,Cell-Signaling) rabbit-anti-Bcl-x_(L)-polyclonal-antibody (1:1000,Santa-Cruz, Heidelberg, Germany) andmouse-anti-β-actin-monoclonal-antibody (1:5000, Sigma). As secondaryantibodies peroxidase-conjugated-goat-anti-mouse IgG orperoxidase-conjugated-goat-anti-rabbit IgG (1:5000, Santa-Cruz) wereused for the enhanced chemoluminescence system (ECL, Amersham-Pharmacia,Freiburg, Germany). Equal protein loading was controlled by β-actindetection.

Analysis of Doxorubicin Uptake and Efflux

For analysis of doxorubicin uptake, the BCP-leukemia cell line Tanouewas seeded in a density of 100,000 cells/mL in 175 cm² flasks and waseither left untreated or incubated with 0.3 μg/mL doxorubicin or acombination of 0.3 μg/mL doxorubicin and 3 μg/mL D,L-methadone at 37°C./5%/CO₂. After 24h, cells were washed twice with ice-cold PBS/1% FCS.Relative doxorubicin uptake in cells was analyzed using flowcytometry.

For analysis of doxorubicin efflux, cells were washed to removedoxorubicin from medium after incubation for 24h. Next, cells wereincubated with fresh medium without doxorubicin or fresh mediumcontaining 3 μg/mL D,L-methadone at 37° C./5%/CO₂ without doxorubicin tomeasure doxorubicin efflux. After different time points cells wereharvested, washed and relative doxorubicin content in leukemia cells wasanalyzed using flowcytometry.

II. EXAMPLES Example 1 D,L-Methadone Induces Cell Death inXenograft-Derived all-Cells Depending on Opioid Receptor Expression

To show the clinical relevance of D,L-methadone in treatment of leukemiaand the role of opioid receptor triggering in cell death induction, theanti-cancer effect of D,L-methadone was analyzed in differentxenograft-derived ALL-cells. The xenografts were originally establishedfrom patients with T-cell (ALL-SCID6, ALL-SCID3), B-cell (ALL-SCID7)(Borgmann et al., 2000) and B-cell precursor (BCP, pre-B-ALL-SCID) acuteleukemia. At first, the opioid-receptor expression onxenograft-derived-ALL-cells was measured. It was observed that theALL-SCID6, ALL-SCID3 and the ALL-SCID7 leukemia cells displayedopioid-receptors in high amounts (FIG. 1A), whereas the pre-B-ALL-SCIDexpressed only moderate levels of opioid-receptors (FIG. 1A).

To analyze if cell death induction using D,L-methadone depends on thelevels of opioid receptor expression, ALL-SCID6, ALL-SCID3, ALL-SCID7and pre-B-ALL-SCID were treated with different concentrations ofD,L-methadone (FIG. 1 B).

Therapeutic plasma concentrations of D,L-methadone (≦3 μg/mL) were usedbut also a higher concentration of 10 μg/mL D,L-methadone was used,because levels of D,L-methadone in lymphatic tissue and marrow may behigher, but have not been measured (Singh et al., 2011). It was foundthat therapeutic plasma concentrations of D,L-methadone (≦3 μg/mL)induced a strong cell death in xenograft-derived ALL-cells expressinghigh amounts of opioid-receptors on their cell surface (FIG. 1A,B). Incomparison to these observations, the pre-B-ALL-SCID having a moderateopioid-receptor level (FIG. 1A) could only be slightly killed withtherapeutic concentrations of D,L-methadone (FIG. 1B). This clearlyreveals that apoptosis induction by D,L-methadone is depend on the levelof opioid-receptor expression.

Example 2 Combination treatment with D,L-Methadone and doxorubicin killsand

activates caspases in ALL-cells with moderate opioid receptor expressionIn analogous studies, the cytotoxic potential of D,L-methadone onBCP-ALL-cell lines (Tanoue, Reh, Nalm6) expressing opioid-receptors in amoderate level on their cell surface (FIG. 2A) was tested.

These BCP-ALL-cell lines could only be killed slightly by D,L-methadone(FIG. 2 b) as observed for the pre-B-ALL-SCID (FIG. 1B). In order toshow if different substances will act synergistically, the cell linesTanoue, Reh, Nalm6 and pre-B-ALL-SCID were treated with differentconcentrations of D,L-methadone and doxorubicin alone or in combinationwith each other (FIG. 2B, 2C). It was observed that the combinationtreatment strongly induced cell kill in BCP-ALL-cell lines as well as inxenograft-derived-BCP-ALL-patient-cells (pre-B-ALL-SCID) (FIG. 2B,C).

In order to analyze the molecular pathways of cell killing in moredetail and to find out how the combination treatment with D,L-methadoneand doxorubicin induced apoptosis, it was analyzed at first whicheffector molecules of apoptosis signaling are activated in BCP-ALL-cellsupon this combination treatment compared to cells treated withD,L-methadone or doxorubicin alone. 120h after treating the BCP-ALL-cellline Tanoue with D,L-methadone in addition to doxorubicin, theactivation of the caspase cascade in BCP-ALL-cells was observed. Theanalysis revealed a strong activation of caspase-3, caspase-9, andcaspase-2 and cleavage of the prototype substrate of caspase-3,poly-(ADP-ribose)-polymerase (PARP) (FIG. 3A).

The role of the caspase cascade in apoptosis induction was furtherinvestigated with the broad-spectrum inhibitor of caspases zVAD.fmk.BCP-ALL-cells were pre-incubated with or without 50 pM of zVAD.fmk andtreated with D,L-methadone in addition to doxorubicin. zVAD.fmk stronglydecreased cell death after combination treatment with D,L-methadone anddoxorubicin in BCP-ALL-cells (FIG. 3B) underlining the dependence oncaspases activation.

The apoptotic machinery is tightly controlled by anti-apoptotic factorslike XIAP and Bcl-x_(L)(Fulda, 2009a; Fulda, 2009b) which we found to bestrongly downregulated in BCP-ALL-cells treated with D,L-methadone inaddition to doxorubicin (FIG. 3C). These results indicate that thecombination of D,L-methadone and doxorubicin sensitizes BCP-ALL-cellsfor apoptosis via the activation of caspases and downregulation of XIAPand Bc1-x_(L).

Example 3 Doxorubicin Strongly Induces Opioid-Receptor Expression inLeukemia Cells

The efficiency of cell death induction and activation of effectormolecules in apoptosis pathways after treating leukemia cells withD,L-methadone seems to depend on the amount of opioid-receptorsdisplayed on the cell's surface. Combination treatment withD,L-methadone and doxorubicin profoundly kills leukemia cells withmoderate opioid receptor expression, which could only be killed slightlyby D,L-methadone or doxorubicin alone. Chemotherapeutics enhance theexpression of receptors like CD95 in leukemia cells (Posovszky et al.,1999). To analyze whether doxorubicin might influence theopioid-receptor expression, the BCP-ALL-cell line Tanoue was treatedwith doxorubicin for 96h. Afterwards, the relative amount ofopioid-receptors compared to untreated cells was measured byflowcytometry. It was found that doxorubicin strongly increasedopioid-receptor expression (FIG. 4A) suggesting that D,L-methadone canbind in higher amounts to cells co-treated with doxorubicin. This effectcould presumably result in the higher cytotoxic potential of thecombination treatment with D,L-methadone and doxorubicin.

Example 4 Opioids Like D,L-Methadone Enhances the Uptake of Doxorubicinand Inhibits its Efflux

Opioids are substrates of the in multi-drug resistances-involved effluxpump P-glycoprotein (P-gp). To analyze whether D,L-methadone mightinfluence the uptake and/or efflux of doxorubicin in leukemia cells, theBCP-ALL-cell line Tanoue was incubated for different intervals withdoxorubicin alone or with a combination of doxorubicin andD,L-methadone. After 24h (Oh), an enhanced doxorubicin concentration inthe cells co-incubated with doxorubicin and D,L-methadone (FIG. 4B) wasobserved. After removing doxorubicin from the supernatant, fresh mediumwas added without doxorubicin and D,L-methadone was applied. After 8hand 24h, D,L-methadone reduced the doxorubicin efflux strongly (FIG. 4B)indicating that D,L-methadone increases doxorubicin uptake and inhibitsdoxorubicin efflux out of leukemia cells. This explains howD,L-methadone as well as doxorubicin mutually increase their cytotoxicpotential.

Example 5 Apoptosis Induction by D,L-Methadone and Doxorubicin DependsCritically on Opioid Receptor Activation and cAMP Concentration

To further analyze the role of opioid-receptor triggering in apoptosisinduction and consequently activation of apoptotic pathways, theBCP-ALL-cell line Tanoue was treated with D,L-methadone, doxorubicin orwith the opioid-receptor antagonist naloxone alone or in differentcombinations with each other (FIG. 5A,B).

After 96h it was found that blocking opioid-receptors by naloxonestrongly reduced the apoptosis rates of the combination treatment withD,L-methadone and doxorubicin (FIG. 5A). In addition, opioid-receptorblocking by naloxone drastically reduced the activation of caspase-9,caspase-2 and caspase-3 and cleavage of PARP after treatingBCP-ALL-cells with D,L-methadone in addition to doxorubicin (FIG. 5B).This indicates that opioid-receptor triggering is critically involved inapoptosis induction and in caspase activation (FIG. 7).

Opioid receptor stimulation activates inhibitory G_(i)-proteins which inturn block adenylyl cyclase activity reducing cAMP (FIG. 7). cAMP is aninhibitor of DNA-damage—as well as doxorubicin-induced apoptosis inleukemia cells (Naderi et al., 2009; Safa et al., 2010a). Pertussistoxin (PTX) inactivates G_(i)-proteins and blocks downregulation of cAMP(Law et al., 1985) (FIG. 7). IBMX however increases cAMP levels as aresult of phosphodiesterase inhibition (FIG. 7). To analyze the criticalrole of cAMP in opioid receptor activation-induced apoptosis, theBCP-ALL-cell line Tanoue was treated with D,L-methadone, doxorubicin,and IBMX or PTX either alone or in different combinations with eachother (FIG. 5C,D). After 96h it was found that upregulation of cAMP byIBMX (FIG. 5C) and blocking downregulation of cAMP by PTX (FIG. 5D)strongly reduced the apoptosis rates of combination treatment withD,L-methadone and doxorubicin. In addition, the upregulation of cAMP byIBMX also decreased doxorubicin-induced apoptosis (FIG. 5C). Theseresults indicate that the activation of opioid receptor coupledG_(i)-proteins is essential for the induction of apoptosis which mightbe regulated via the intracellular cAMP levels.

Example 6 D,L-Methadone Alone or in Addition to Doxorubicin InhibitsTumour Growth In Vivo in an ALL-Xenograft-Model

In vitro results demonstrated that D,L-methadone could induce apoptosisin several leukemia cell lines and increased the cytotoxicity ofdoxorubicin. To confirm the clinical relevance of the anti-cancerpotential of D,L-methadone alone or in combination with doxorubicin andto verify the results obtained so far an ALL-xenograft study wasundertaken.

For the in vivo study, a patient-derived-ALL-xenograft-model (ALL-SCID6)was used. Its phenotypic and genotypic identity with the originalpatient sample was proven (Borgmann et al., 2000). The experimentstarted at day 0 with subcutaneous inoculation of ALL-SCID6 fragmentsfrom an in vivo passage into male NOD/SCID/IL2ry null (NSG) mice. Afterrandomization, D,L-methadone was orally administered starting at day oneafter ALL-inoculation with increasing doses. When tumours were palpable,doxorubicin treatment was initiated. D,L-methadone and doxorubicintreatment led to a significant inhibition of tumour growth at comparablelevels (FIG. 6).

Combination treatment with D,L-methadone and doxorubicin had a similaranti-tumour efficacy as D,L-methadone or doxorubicin alone until day 70.At later time points, the tumour inhibition was longer lasting duringthe combined treatment of D,L-methadone and doxorubicin. The therapy waswell-tolerated with body weight changes of −10% for the combination and−8% or −4% for the D,L-methadone or doxorubicin treatment, respectively.To analyze D,L-methadone serum concentrations in mice, 0.5, 1, 4 and 24hours after the last D,L-methadone application, serum was taken andD,L-methadone quantified by mass spectrometry. The serum concentrationsof methadone were found between 28 ng/mL and 138 ng/mL in the timecourse of 0.5 until 4 hours after D,L-methadone application indicatingthat levels comparable with the in vitro concentrations could bereached. The serum concentrations of doxorubicin were found between 156ng/mL and 198 ng/mL. These results demonstrate that D,L-methadone andthe co-treatment using doxorubicin and D,L-methadone significantlyinhibited tumour growth in vivo.

Example 7 D,L-Methadone Sensitizes Glioblastoma Cells for DoxorubicinTreatment

As shown by flow cytometry, the glioblastoma cell lines A172 and U118MG(s. FIG. 8) as well as primary glioblastoma cells (s. FIG. 11A) andglioblastoma-initiating stem cells (s. FIG. 12A) express opioidreceptors. In all these cells and cell lines the combination treatmentof D,L-methadone and doxorubicin dose-dependently induces apoptosis (seeFIGS. 9, 10, 11B and 12B). As exemplified for the glioblastoma cell lineA172 it could be shown that cell death induction of glioma cells usingD,L-methadone and doxorubicin cotreatment depends on caspase activation(s. FIG. 13). Furthermore, it could be shown that D,L-methadone reverseddeficient activation of apoptosis pathways by doxorubicin inglioblastoma-initiating stem cells (s. FIG. 14).

Example 8 Effect of D,L-Methadone on Doxorubicin Uptake and Efflux

In vitro results using the glioblastoma cell line A172 demonstrated thatD,L-methadone could enhance the uptake and also inhibit the efflux ofdoxorubicin (s. FIG. 15). This gives an explanation for thesensitization of cancer cell towards treatment with anticancer drugs.

Example 9 Effect of Doxorubicin or Cisplatin on Opioid ReceptorExpression on Cancer Cells

As shown for the glioblastoma cell line A172, doxorubicin leads to a6-fold increase in opioid receptor expression (s. FIG. 16). It could beshown that this mechanism holds also true for other cancer types andanticancer drugs since in the promyelocytic leukemia cell line HL60, thecisplatin-treatment leads to a 2.1-fold increase in opioid receptorexpression (s. FIG. 19).

Example 10 Sensitization of leukemia, pancreatic and ovarian cancercells for

treatment with different anticancer agents. In further in vitro analysesit could be demonstrated that D,L-methadone sensitizes leukemia cancercells (Nalm-6), pancreatic cancer cells (Nalm6) and ovarian cancer cells(A2780) for etoposide or cisplatin treatment (s. FIG. 17). Furthermore,also chronic lymphocytic leukemia cells (CLL) could be sensitized byD,L-methadone for apoptotic treatment using Fludarabine (s. FIG. 18).

Example 11 Sensitization of mamma carcinoma cells for treatment withdifferent

anticancer agents. As shown by flow cytometry, the Her2/Neu-resistentmamma carcinoma cell line JIMT-1 expresses the p-opioid receptor (s.FIG. 20). As shown by FACS analysis the combination treatment ofD,L-methadone and doxorubicin dose-dependently induces apoptosis inJIMT-1 cells (see FIG. 21). It could be shown that cell death inductionof JIMT-1 cells using D,L-methadone and doxorubicin cotreatment dependson caspase activation (s. FIGS. 22 and 23).

Example 12 Sensitization of cancer cells for doxorubicin treatment bythe opioid

fentanyl. As exemplified for the T-cell derived leukemia cell line CEMit could be shown that also the opioid fentanyl was able to sensitizethe CEM cells for treatment using doxorubicin (s. FIG. 24). In a furtherin vitro experiment, the opioid buprenorphine sensitized leukemia cells(HL-60) for apoptosis due to doxorubicin (s. FIG. 25).

Example 13 Combination Treatment with D,L-Methadone and Cisplatin (CDDP)Kills and Activates Caspases in Different Leukemia Cells

The cell death potential of D,L-methadone on different leukemia-celllines was shown on human T cell leukemia, human acute myeloid leukemia,human B cell precursor leukemia, human B cell leukemia. All tested celllines expressing opioid-receptors in a moderate level on their cellsurface (FIG. 27).

These leukemia cells could only be killed slightly by D,L-methadone(FIG. 28, white bars). In order to show that different anticancer agents(substances) will act synergistically, human T cell leukemia, humanacute myeloid leukemia, human B cell precursor leukemia and human B cellleukemia were treated with different concentrations of D,L-methadone(−CDDP) or cisplatin alone or with D,L-methadone in addition tocisplatin (+CDDP) (FIG. 28). The combination treatment strongly inducedcell death in different leukemias depending on different concentrationsof cisplatin or/and D,L-methadone.

The molecular pathways of cell killing was shown in more detail and itwas shown how the combination treatment with an opioid receptor agonisti.e. D,L-methadone and an anticancer agent i.e. cisplatin induceapoptosis. First the effector molecules of apoptosis signaling wasshown, that are activated in different leukemia cells (human T cellleukemia, human acute myeloid leukemia, human B cell precursorleukemia). Combination treatment of D,L methadone in combination withCisplatin (+CDDP) was compared to cells treated with D,L-methadone(−CDDP) or cisplatin alone. It was shown that treating the differentleukemia cells with D,L-methadone in addition to cisplatin (+CDDP), theactivation of the caspase cascade in leukemia cells was induced. Astrong activation of caspase-3 (active caspase-3 p19, p17), caspase-9(active caspase-9 p37), and caspase-2 and cleavage of the prototypesubstrate of caspase-3, poly-(ADP-ribose)-polymerase (PARP) (cleavagep85 and or a downregulation of PARP p116) was induced depending on thecombination treatment (FIG. 29).

The role of the caspase cascade in apoptosis induction was furtherinvestigated with the broad-spectrum inhibitor of caspases zVAD.fmk.Different leukemia cells (human T cell leukemia, human acute myeloidleukemia, human B cell precursor leukemia) were pre-incubated with 50 μMof zVAD.fmk (+zVAD.fmk, white bars) or without zVAD.fmk (−zVAD.fmk,black bars) and treated with D,L-methadone in addition to cisplatin.zVAD.fmk strongly decreased cell death after combination treatment withD,L-methadone and cisplatin (FIG. 30) underlining the dependence oncaspases activation.

The apoptotic machinery is tightly controlled by anti-apoptotic factorslike XIAP and Bcl-xL and pro-apoptotic factors like Bax (Fulda, 2009a;Fulda, 2009b). XIAP was strongly downregulated (p57) and or cleaved(p30) in different leukemia cells treated with D,L-methadone in additionto cisplatin (+CDDP) (FIG. 29) depending on different concentrations ofcisplatin or/and different concentration of D,L-methadone. A strongupregulation of Bax (p21) is induced in human T cell leukemia inducedafter treatment with D,L-methadone in addition to cisplatin (+CDDP). Thecombination of D,L-methadone and cisplatin sensitizes different leukemiacells for apoptosis via the activation of caspases and by downregulationand inhibition of anti-apoptotic factors such as XIAP and upregulationof pro-apoptotic factors such as Bax. So it shown that the opioidreceptors are receptors which induce cell death and activate apoptosispathways involving caspase activation, downregulation and or cleavage ofPARP, and or downregulation of anti-apoptotic factors, and orupregulation of pro-apoptotic factors, and or downregulation andinhibition of inhibitory apoptotic proteins (IAP). Therefore the opioidreceptors are a new unknown way with an new mechanism of inducing celldeath, beside the common known cell death receptors/death inducingligands systems and mechanisms like the CD95/CD95L-System.

Example 14 Cisplatin Strongly Induces Opioid-Receptor Expression inLeukemia Cells

The efficiency of cell death induction and activation of effectormolecules in apoptosis pathways after treating leukemia cells withopioid-receptor-agonists i.e. D,L-methadone depend on the amount ofopioid-receptors displayed on the cell's surface. Combination treatmentwith D,L-methadone and cisplatin profoundly kills leukemia cells withmoderate opioid receptor expression, which could only be killed slightlyby D,L-methadone or cisplatin alone. Chemotherapeutics enhance theexpression of the receptor CD95 (FAS,APO-1) in leukemia cells which is aspecial known death receptor (Posovszky et al., 1999). To show thatcisplatin has an influence to the opioid-receptor expression, thedifferent leukemia cells (human T cell leukemia, human acute myeloidleukemia, human B cell precursor leukemia) were treated with cisplatin.Afterwards, the relative amount of opioid-receptors compared tountreated cells was measured by flowcytometry. It was shown thatcisplatin strongly increased opioid-receptor expression (FIG. 31).Therefore opioid receptor-agonists like D,L-methadone can bind in higheramounts to cells, co-treated with cisplatin or other anticancer agentswhich are able to induce a higher level of expressed opioid receptors.This effect results in the higher cell death potential of thecombination treatment of an opioid receptor agonist i.e D,L-methadoneand an anticancer agent i.e. cisplatin.

It is shown in the invention that the opioid receptor agonist which hasa longer minimal duration of effectiveness like D,L methadone has abetter result than one, that has a shorter minimal duration ofeffectiveness like morphine compared to D,L methadone (FIG. 33 and FIG.34.

Example 15 Apoptosis Induction by D,L-Methadone and Doxorubicin DependsCritically on Opioid Receptor Activation in Glioblastomas

To show the role of opioid-receptor triggering in apoptosis induction inglioblastomas which is a solid tumour, glioblastoma cells were treatedwith D,L-methadone, doxorubicin or with the opioid-receptor antagonistnaloxone alone or in different combinations with each other (FIG. 32).After 120h and 144h it was shown that blocking opioid-receptors bynaloxone strongly reduced the apoptosis rates of the combinationtreatment with D,L-methadone and doxorubicin (FIG. 32).

Example 16 Different Duration of Effectiveness of Different OpioidsInduces Different Rates of Apoptosis Using D,L-Methadone or Morphine inCombination with Anticancer Drugs Such as Doxorubicin in Glioblastomasor in Leukemias

The efficiency of cell death induction after treating glioblastoma cellsor leukemia cells with opioids depends on the duration of effectivenessof the opioids. The minimal duration of effectiveness of methadone is5-7 hours and the minimal duration of effectiveness of morphine is 2-4hours. Combination treatment with D,L-methadone and doxorubicin stronglyinduced high cell death rates in glioblastoma cells (FIG. 33 A) andleukemia cells (FIG. 34 A). In contrast, combination treatment withmorphine and doxorubicin induced lower cell death rates in glioblastomacells (FIG. 33 B) and leukemia cells (FIG. 34 B). This indicates thatthe rates of induction of cell death after combination treatment ofopioids with anticancer drugs depend also on the duration ofeffectiveness of opioids. The effect is found also at other anticanceragents.

Example 17 Combination Treatment Using D,L-Methadone in Addition toDoxorubicin

Mediated Cell Proliferation Inhibition and G2/M Cell Cycle Arrest inGlioblastoma Cells. Cell proliferation is governed by the eukaryoticcell cycle (Sherr C J. Cancer cell cycles. Science 1996; 274:1672-7),which is regulated not only by growth factors but also by a variety ofsignals that act to inhibit cell cycle progression. Most of cancer cellshave 4 cell division cycle stages: gap 1 (G1), synthesis (S), G2, andmitosis (M). Chromosomal DNA replicates during the S phase. Asglioblastoma cells divide, the cell cycle should move from the S stageto the G2/M stage. This tightly controlled temporal order is imposed bythe sequential activation of a number of protein kinases known ascyclin-dependent kinases (CDKs), by the formation of complexes withvarious cyclins. Opioid receptor agonist for example Methadone incombination with doxorubicin inhibits proliferation of cancer cells suchas glioblastoma cells and induces S/G2-M cell cycle arrest inglioblastoma cells.

Example 18 Apoptosis Induction and Caspase Activation Depend on OpioidReceptor Activation Inducing cAMP Downregulation in Glioblastoma

cAMP-related signaling can control apoptosis induction and cell growth.To analyze the role of opioid receptor activation in apoptosis inductionand caspase activation in glioblastoma cells, glioblastoma cells A172were treated with the opioid receptor agonist D,L-methadone, theanticancer agent doxorubicin or with the opioid receptor antagonistnaloxone alone or in different combinations (FIGS. 36A, 36B and 36C).Blocking opioid receptors by naloxone strongly reduced apoptosis (FIGS.36A and 36B) and activation of caspase-9, caspase-3 and cleavage of PARP(FIGS. 36C) induced by combination treatment with D,L-methadone anddoxorubicin. This indicates that opioid receptor activation plays acritical role in apoptosis induction and caspase activation. Opioidreceptor stimulation activates inhibitory Gi-proteins which in turnblock adenylyl cyclase activity reducing cAMP. IBMX, however, increasescAMP levels due to phosphodiesterase inhibition. To analyze the criticalrole of cAMP in opioid receptor activation-induced apoptosis, A172 cellswere treated with D,L-methadone, doxorubicin, and IBMX either alone orin different combinations (FIG. 36D). Upregulation of cAMP by IBMXstrongly reduced apoptosis induction by combination treatment withD,L-methadone and doxorubicin indicating that opioid receptor activationvia cAMP downregulation sensitizes glioblastoma cells fordoxorubicin-induced apoptosis and caspases activation.

Example 19 Opioid Receptor Activation Using D,L-Methadone InhibitsTumour Growth In Vivo

U87MG glioblastoma cells were subcutaneously inoculated per nude-mouse.After randomization of 16 mice, D,L-methadone was daily orallyadministered in 8 mice starting at day 1 until the end of experiment.D,L-Methadone dosage was increased weekly from 60 to 120 to 240 mg/kg/dbid. At day 33, 24h after the last treatment with D,L-methadone the micewere sacrificed. For analyzing serum concentrations of D,L-methadone inmice 0.5, 1 and 4h after last D,L-methadone-application, serum was takenand D,L-methadone quantified by mass spectrometry. In comparison tovehicle treated mice of the control group, the D,L-methadone treatedmice had a significantly reduced tumour size at day days 19 to 33 withan optimum T/C value of 49% (see FIG. 37). The D,L-methadone treatmentwas well tolerated in the dose used and induced only a minor body weightloss of 9%. Serum concentrations were found between 136 ng/ml and 1608ng/mL of methadone in the time course of 0.5 to 4h after D,L-methadoneapplication. These findings demonstrate that opioid receptor activationusing D,L-methadone inhibits growth of glioblastoma in vivo.

Example 20 Opioids Such as D,L-Methadone Increase Cisplatin-Induced CellDeath in Ovarian Cancer Cell after Short Term Treatment

A2780 ovarian cancer cells were treated with cisplatin (5, 3 μg/mL) orD,L-methadone (3, 1 μg/mL) alone or in combination. As shown in FIG. 38,a strong induction of cell death was observed by co-treatment ofD,L-methadone and cisplatin. This suggests that opioids such asD,L-methadone strongly potentiates cisplatin-induced apoptosis inovarian cancer cells.

Example 21 Opioids Such as D,L-Methadone Increase Cisplatin-Induced CellDeath in Ovarian Cancer Cell after Long Term Treatment

A2780 ovarian cancer cells were treated with cisplatin (2, 1, 0.5, 0.3μg/mL) or D,L-methadone (10, 3, 1 μg/mL) alone or in combination. Asshown in FIG. 39, a strong induction of cell death was observed byco-treatment of D,L-methadone and cisplatin. This suggests that opioidssuch as D,L-methadone strongly potentiates cisplatin-induced apoptosisin ovarian cancer cells and breaks chemoresistance.

Example 22 Opioids Such as D,L-Methadone Increase Cisplatin-Induced CellDeath in Cisplatin-Resistant Ovarian Cancer Cell

A2780cis ovarian cancer cells were treated with cisplatin (3, 2, 1μg/mL) or D,L-methadone (10, 3, 1 μg/mL) alone or in combination. Asshown in FIG. 40, a strong induction of cell death was observed byco-treatment of D,L-methadone and cisplatin. This suggests that opioidssuch as D,L-methadone strongly potentiates cisplatin-induced apoptosisin cisplatin-resistant ovarian cancer cells.

Example 23 Opioids Such as D,L-Methadone Increase the Effectiveness ofCisplatin in Treatment of Ovarian Cancer Cell

A2780 ovarian cancer cells were treated with cisplatin (2 μg/ml) alone.As shown in FIG. 41, cisplatin in a concentration of 2 μg/mL inducedcell death of 90% after 144h. However, treatment with 0.5 μg/mLcisplatin in addition to D,L-methadone (10, 3, 1 μg/mL) induced a celldeath of 95%. Furthermore, treatment with 0.2 μg/mL cisplatin inaddition to D,L-methadone (10, 3, 1 μg/mL) induced a cell death ofbetween 70 and 85% depending on concentrations of D,L-methadone. Thisindicates that opioids such as D,L-methadone strongly potentiatescisplatin-induced apoptosis/cell death in ovarian cancer cells.D,L-methadone increases the effectiveness of cisplatin in treatment ofovarian cancer, suggesting that a strong reduction of anti-cancer drugsconcentrations can be used by cotreatment with D,L-methadone to getcomparable cell death rates and therefore less side effects of theanti-cancer drugs will be observed. Based on the present data, theanticancer agent can be given at a dose which it at least 2 or 3 timeslower and up to 100 times lower than the recommended dose for thetreatment of the respective cancer.

Example 24 Opioid Receptor Activation Reverses Deficient CaspaseActivation by Cisplatin in Ovarian Cancer Cells

Deficient caspase activation was observed in chemo- and radio resistantovarian cancer cells treated with anticancer drugs or radiation. Toclarify the involvement of caspases activation in combination therapy ofD,L-methadone and cisplatin-induced apoptosis in ovarian cancer cells,A2780 ovarian cancer cells were treated with D,L-methadone (3, 1 μg/mL)or cisplatin (5, 3 μg/mL) alone or in combination. As shown in FIG. 42,the combination treatment using cisplatin and D,L-methadone leads to astrong caspase activation in ovarian cancer cells by activatingcaspase-3, caspase-9, and caspase-8 and cleavage ofPoly-(ADP-ribose)-polymerase (PARP). This demonstrates that opioidreceptor activation using D,L-methadone reverses deficient activation ofcaspases by cisplatin in ovarian carcinoma cells.

Example 25 Opioid Receptor Activation Using D,L-Methadone Plays aCritical Role in Sensitizing Breast Cancer Cells for DoxorubicinTreatment

To analyze the role of opioid receptor activation in apoptosis inductionin breast cancer, the Trastuzumab resistant breast cancer cells JIMT-1were treated with the opioid receptor agonist D,L-methadone, doxorubicinor with the opioid receptor antagonist naloxone alone or in differentcombinations (FIG. 43). Blocking opioid receptors by naloxone stronglyreduced apoptosis induced by combination treatment with D,L-methadoneand doxorubicin. This indicates that opioid receptor activation plays acritical role in apoptosis induction.

Example 26 Opioids Such as D,L-Methadone Increase Cisplatin-Induced CellDeath in Prostate Cancer Cells

Prostate cancer cells PC-3 were treated with cisplatin (5, 3 μg/mL) orD,L-methadone (10, 3, 1 μg/mL) alone or in combination. As shown in FIG.44, a strong induction of cell death was observed by co-treatment ofD,L-methadone and cisplatin. This suggests that opioids such asD,L-methadone strongly potentiates cisplatin-induced apoptosis inprostate cancer cells.

Example 27 Opioids Such as D,L-Methadone Increase Cell Death Inductionof Different Anti-Cancer Drugs from Different Anti-Cancer Drug Classesin Leukemia Cells

Leukemia cells Nalm6 were treated with different anti-cancer drugs alone(white columns) or in combination with D,L-methadone (black columns). Asshown in FIG. 45, a strong induction of cell death was observed byco-treatment of D,L-methadone and anti-cancer drugs (black columns).This suggests that opioids such as D,L-methadone strongly potentiatesapoptosis induction of different anticancer drugs from differentanticancer classes in leukemia cells.

Example 28 Opioids Such as D,L-Methadone Increase Anti-CancerDrug-Induced Cell in Glioblastoma Cells

Glioblastoma cells A172 were treated with different anti-cancer drugsfrom the same anti-cancer drug class such as anthracyclines(Doxorubicin, Idarubicin, and Daunorubicin). Glioblastoma cells weretreated with anthracyclines alone (white columns) or in combination withD,L-methadone (black columns). As shown in FIG. 46, a strong inductionof cell death was observed by co-treatment of D,L-methadone anddifferent anthracyclines. This suggests that opioids such asD,L-methadone strongly potentiates apoptosis induction of differentanti-cancer drugs from the same class in glioblastoma cells.

Example 29 Opioid Receptor Expression on Pancreatic Cancer Cells

Pancreatic cancer cells Colo 357 were stained with naloxone fluoresceinmeasuring opioid receptor expression by flow cytometry. As a result astrong expression of opioid receptors on the surface of pancreaticcancer was found (see FIG. 47).

Example 30 Opioids Such as D,L-Methadone Increase Anti-CancerDrug-Induced Cell in Pancreatic Cancer Cells

Pancreatic cancer cells Colo 357 were treated with different anti-cancerdrugs from the same anti-cancer drug class such as cisplatin metalcomplexes (oxaliplatin, cisplatin). Pancreatic cancer cells Colo 357were treated with different concentration of cisplatin metal complexes,oxaliplatin or cisplatin alone or in combination with D,L-methadone. Asshown in FIG. 48, a strong induction of cell death was observed byco-treatment of D,L-methadone and the platin metal complexes (A)oxaliplatin or (B) cisplatin. This suggests that opioids such asD,L-methadone strongly potentiates apoptosis induction of differentanticancer drugs from the same anti-cancer drug class in pancreaticcancer cells.

Example 31 Opioid Receptor Activation Reverses Deficient CaspaseActivation by Cisplatin and Oxaliplatin in Pancreatic Cancer Cells

Deficient caspase activation was observed in chemo- and radioresistantpancreatic cancer cells Colo 357 treated with anti-cancer drugs orradiation. To clarify the involvement of caspases activation incombination therapy of D,L-methadone and cisplatin-induced apoptosis orD,L-methadone and oxaliplatin-induced apoptosis in pancreatic cancer,pancreatic cancer cells Colo 357 were treated with D,L-methadone (3, 1μg/mL) or oxaliplatin (3, 2 μg/mL; see FIG. 49A) or cisplatin (0.5, 0.7μg/mL; see FIG. 49 B) alone or in combination with methadone andoxaliplatin (see FIG. 49A) or cisplatin (see FIG. 49B). As shown in FIG.49, the combination treatment leads to strong caspase activation inpancreatic cancer cells by activation of caspase-3, caspase-9, andcleavage of Poly-(ADP-ribose)-polymerase (PARP). This demonstrates thatopioid receptor activation using D,L-methadone reverses deficientactivation of caspases by cisplatin or oxaliplatin in pancreatic cancercells.

Example 32 Inhibition of Caspases Activation Blocks Opioid-SensitizedPancreatic Cancer Cells for Oxaliplatin- or Cisplatin-Induced Apoptosis

To investigate the critical role of caspases in opioid receptoractivation-induced apoptosis, pancreatic cancer cells Colo 357 wereincubated with the broad spectrum inhibitor of caspases zVAD.fmk.Incubation with zVAD.fmk almost completely inhibited apoptosis inpancreatic cancer cells induced by D,L-methadone in addition tooxaliplatin (see FIG. 50 A,B) or by D,L-methadone in addition tocisplatin (see FIG. 50 C,D), suggesting that caspases are central foropioid receptor activation-mediated sensitization of pancreatic cancercells for oxaliplatin and cisplatin treatment. This demonstrates thatopioid receptor activation such as D,L-methadone reverses deficientactivation of caspases by oxaliplatin or cisplatin in pancreatic cancercells.

Example 33 Opioids Such as D,L-Methadone Increase Temozolomide(Temodal)—Induced Cell Death in Glioblastoma Cells

Glioblastoma cells A172 were treated with temozolomide or D,L-methadone(3, 1 μg/mL) alone or in combination. As shown in FIG. 51, a stronginduction of cell death was observed by co-treatment of D,L-methadoneand temozolomide. This suggests that opioids such as D,L-methadonestrongly potentiates temozolomide-induced apoptosis in glioblastomacells.

Example 34 Opioids Such as D,L-Methadone Increase the Effectiveness ofOxaliplatin and Cisplatin in Pancreatic Cancer Treatment

Pancreatic cancer cells Colo 357 were treated with differentconcentrations of oxaliplatin (A) or cisplatin (B) alone or incombination with D,L-methadone (hatched columns, white columns). Asshown in FIG. 52, a strong induction of cell death was observed byco-treatment of D,L-methadone and different cisplatin metal complexesoxaliplatin (A) or cisplatin (B).

Pancreatic cancer cells Colo 357 were treated with (A) oxaliplatin (10μg/ml) alone. 10 μg/mL cisplatin induced cell death of 60% after 120h.However, treatment with 3 μg/mL oxaliplatin in addition to D,L-methadone(10, 3, 1 μg/mL) induced a cell death of 65%. In addition, treatmentwith 2 μg/mL oxaliplatin in addition to D,L-methadone (10, 3, 1 μg/mL)induced a cell death of 45%.

Pancreatic cancer cells Colo 357 were treated with (B) cisplatin (10μg/ml) alone. 10 μg/mL cisplatin induced cell death of 70% after 144h.However, treatment with 0.7 μg/mL cisplatin in addition to D,L-methadone(10, 3, 1 μg/mL) induced a cell death of 85%. In addition, treatmentwith 0.5 μg/mL cisplatin in addition to D,L-methadone (10, 3, 1 μg/mL)induced a cell death of 60%.

This suggests that D,L-methadone increases the effectiveness ofcisplatin or oxaliplatin in treatment of pancreatic cancer, suggestingthat a strong reduction of anti-cancer drugs concentrations can be usedby cotreatment with D,L-methadone to get comparable cell death rates andtherefore less side effects of the anti-cancer drugs will be observed.In addition, this demonstrates that opioids such as D,L-methadone breakschemoresistance because conventional therapies using anti-cancer drugsare limited by the toxicity of anti-cancer drugs concentrations used forpatients treatment.

Example 35 Opioids Such as D,L-Methadone Increase the Effectiveness ofDoxorubicin in Leukemia Treatment

BCP-ALL cell lines (Nalm6, Reh and Tanoue) were treated with doxorubicinalone or in combination with D,L-methadone and doxorubicin (hatchedcolumns). As shown in FIG. 53, a strong induction of cell death wasobserved by co-treatment using D,L-methadone. This suggests thatD,L-methadone increases the effectiveness of doxorubicin in treatment ofleukemia cells, suggesting that a strong reduction of anti-cancer drugsconcentrations can be used by cotreatment with D,L-methadone to getcomparable cell death rates and therefore less side effects of theanti-cancer drugs will be observed.

Example 36 Opioids such as D,L-methadone increase the effectiveness ofDoxorubicin in Breast Cancer Treatment

Trastuzumab resistant breast cancer cells (JIMT-1) were treated withdoxorubicin alone or in combination with D,L-methadone and doxorubicin(hatched columns). As shown in FIG. 54, a strong induction of cell deathwas observed by co-treatment of D,L-methadone. 0.1 μg/mL doxorubicininduced cell death of 70% after 120 h. However, treatment with 0.015μg/mL doxorubicin in addition to D,L-methadone (10, 3, 1, 0.1 μg/mL)induced a cell death between 70 and 55% depending on D,L-methadoneconcentration.

This suggests that D,L-methadone increases the effectiveness ofdoxorubicin in treatment of breast cancer cells, suggesting that astrong reduction of anti-cancer drugs concentrations can be used bycotreatment with D,L-methadone to get comparable cell death rates andtherefore less side effects of the anti-cancer drugs will be observed.

Example 37 Opioids Such as D,L-Methadone Increase the Effectiveness ofDoxorubicin in Glioblastoma Treatment

Glioblastoma cells A172 were treated with different concentrations ofdoxorubicin alone or in combination with D,L-methadone (hatched columns,white columns). As shown in FIG. 55, a strong induction of cell deathwas observed by co-treatment of D,L-methadone and doxorubicin.

Glioblastoma cells A172 were treated with doxorubicin (1 μg/ml) alone. 1μg/mL doxorubicin induced cell death of 80% after 144 h. However,treatment with 0.1 μg/mL doxorubicin in addition to D,L-methadone (10,3, 1 μg/mL) induced a cell death of 85%-50% depending on concentrationsof D,L-methadone.

This suggests that D,L-methadone increases the effectiveness ofdoxorubicin in treatment of glioblastoma, suggesting that a strongreduction of anti-cancer drugs concentrations can be used by cotreatmentwith D,L-methadone to get comparable cell death rates and therefore lessside effects of the anti-cancer drugs will be observed. In addition,this demonstrates that opioids such as D,L-methadone breakschemoresistance because conventional therapies using anti-cancer drugsare limited by the toxicity of anti-cancer drugs concentrations used forpatients treatment.

Example 38 Combination Treatment of Different Opioids Shows aSynergistic Effect for Induction of Cell Death in Leukemia Cells

Leukemia cells HL60 were treated with fentanyl (3, 1 μg/mL) alone (A) ormorphine (3, 1 μg/mL) alone (A) or in combination of fentanyl andmorphine (B) at concentrations as indicated. As shown in FIG. 56, astrong synergistically increased induction of cell death was observed byco-treatment of morphine and fentanyl (B). This suggests that thecombination of different opioids enhances the pro-apoptotic effect andargues for a combined use of opioids also in the combination with afurther anticancer agent.

DISCUSSION

The examples provide evidence that D,L-methadone induces apoptosis,activates caspases and increases doxorubicin-induced cell death inleukemia cells depending on opioid-receptor activation inducing thedownregulation of cAMP. In addition, it could be demonstrated for thefirst time, that D,L-methadone can strongly reduce tumour growth of ALLin a xenograft-model in vivo. Noticeably, this tumour-killing effectcould be enhanced by the combination of D,L-methadone with theanticancer drug doxorubicin.

Methadone is a p-opioid receptor agonist binding to p-opioid receptorsif presented on cells. It was found that D,L-methadone kills stronglyxenograft-derived ALL-cells expressing high levels of opioid receptors.In contrast, D,L-methadone induces cell death only slightly inxenograft-derived ALL-cells and -cell lines expressing moderate opioidreceptor amounts indicating that D,L-methadone-induced apoptosis seemsto depend on critical levels of opioid receptor expression in leukemiacells.

Combination treatment may prove to be advantageous in malignancies thatstill partially respond to either treatment alone as differenttherapeutics are known to interact with each other amplifying weakerdeath signals. Combination treatment with D,L-methadone and doxorubicinenhances the anti-tumour efficacy of both agents synergistically inBCP-ALL-cells expressing moderate levels of opioid-receptors andincreases strongly caspase activation playing a critical role inapoptosis induction in sensitive and resistant cancer cells (Fulda,2009c). In addition, the downregulation of the anti-apoptotic proteinsXIAP and Bc1-x_(L)involved in the occurrence of resistances in manymalignancies like ALL or NHL (Addeo et al., 2005) is markedly enhanced.This suggests that combination treatment of D,L-methadone anddoxorubicin strongly increases apoptosis induction and could improvetheir anti-tumour efficacy synergistically.

Resistance to conventional chemotherapeutic drugs is a limiting factorin the effectiveness of therapies whereby multidrug resistances as aresult of the overexpression of drug transporters such as P-gp are alsowell-characterized. While in healthy cells the P-gp expression belongsto the normal cellular defense system, in human cancer cells theoverexpression of P-gp correlates with decreased survival and pooroutcome (Diestra et al., 2003). D,L-methadone could be shown to be asubstrate of P-gp inhibiting its action (Crettol et al., 2007). As shownherewith, co-treatment of doxorubicin with D,L-methadone enhancesdoxorubicin cell-uptake and furthermore inhibits doxorubicin-efflux outof leukemia cells, suggesting that D,L-methadone sensitizes leukemiacells for doxorubicin-induced apoptosis by increasing concentrations ofdoxorubicin within the cells.

Combination treatment using D,L-methadone and doxorubicin inducedapoptosis and caspase activation in BCP-ALL-cells expressing moderateamounts of opioid-receptors on their surface. The enhanced toxicity ofthis combination treatment was found to be additionally associated withan increased expression of opioid-receptors after doxorubicin treatment.Therefore, D,L-methadone can bind in higher amounts to cells co-treatedwith doxorubicin. These results indicate that the enhanced toxicity inthe combination treatment with D,L-methadone and doxorubicin isassociated with the upregulation of opioid-receptor expression mediatedby doxorubicin and furthermore with an increased uptake and decreasedefflux of doxorubicin mediated by D,L-methadone. Both agents can henceexert their cytotoxic potential to a higher extent.

Opioid receptors signal by catalysing ligand-dependent nucleotideexchange on G_(i), thereby inhibiting adenylyl cyclase and modulatingN-type calcium channels as well as G protein—gated inwardly rectifyingpotassium (GIRK)-type potassium channels leading to changes in cellsignalling (FIG. 7). Dependence of apoptosis induction onopioid-receptor triggering is underlined by their inhibition. Blockingopioid-receptor signaling with the opioid receptor antagonist naloxoneinhibited combination treatment with D,L-methadone anddoxorubicin-induced apoptosis and caspase activation in a high rate,suggesting that opioid-receptor triggering by D,L-methadone is involvedin apoptosis induction and caspase activation (FIG. 7). Based on thismechanism of action every opioid receptor agonist independent of theindividual opioid receptor should kill tumour cells by apoptosis sinceall opioid receptors are linked to the adenylyl cyclase via the G_(i)pathway.

Further experiments prove the general applicability of the abovedescribed combination therapy:

Broad spectrum of cancers. Several diverse cancer types can be treatedwith the combination of opioid receptor agonists such as e.g. breastcancer, pancreatic cancer, prostate cancer, ovarian cancer, glioblastomaor leukemia.

Broad spectrum of opioids. In accordance with the Gi-associatedmechanism of action, several structurally and pharmacologically distinctopioids like D,L-methadone, buprenorphine and fentanyl could sensitizethe cancer cells for anticancer drugs.

Broad spectrum of anticancer drugs. For several structurally andpharmacologically distinct anticancer drugs it could be shown that theyincrease opioid receptor expression and show increased influx/decreasedefflux due to the co-applied opioid agonist.

SUMMARY

It has to be emphasized that the interaction between opioids andanticancer agents represents a self-reinforcing feedback loop asillustrated by FIG. 26. In the first path of this loop opioids enhancethe cellular uptake and inhibit the efflux of anticancer drugs. On thesecond path of said loop the accumulating anticancer drugs lead to anincreased expression of opioid receptors. Hence, both agents can exerttheir cytotoxic potential to a higher extent.

The present examples could verify the clinical relevance withpatient-derived ALL-cells, patient-derived glioblastoma cells andglioblastoma initiating stem cells ex vivo and could show for the firsttime that D,L-methadone as monotherapy or in combination withdoxorubicin leads to a strong tumour growth inhibition in apatient-derived leukemia model and in a glioblastoma xenograft model.The anti-leukemic efficacy, the tumour growth inhibition of glioblastomaand the side effects of D,L-methadone alone or in combination withdoxorubicin were comparable with those of doxorubicin alone. However,only the combination treatment was able to achieve a longer lastinggrowth inhibition. The serum concentrations of methadone in micecorrelated with the concentrations showing in vitro cytotoxicity.

In sum, a combination therapy of opioids and anticancer drugs couldimprove the cancer therapies in several ways:

-   -   Due to the upregulation of opioid receptors, former opioid        insensitive cancer types could be subjected to an opioid        therapy.    -   Due to the opioid-induced intracellular accumulation of        anticancer drugs the efficacy of the treatment is enhanced.    -   Due to inhibition of anti-apoptotic proteins such as Bcl-X_(L)        and inhibition of inhibitory apoptotic proteins such as XIAP    -   This could lead to therapy of cancer types which are difficult        to treat.    -   Furthermore, this might allow a dose reduction for the        anticancer drugs enhancing the safety and patient compliance of        the chemotherapy.    -   Finally, also resistant cancer cells could be re-sensitized for        an anticancer treatment.    -   In addition, the numerous opioids and numerous anticancer drugs        on the market open up the way for new drug combinations which        might represent improved treatment due to increased efficacy        and/or safety.

FIGURES

FIG. 1: D,L-methadone kills ALL cells ex vivo depending on criticallevels of opioid receptor expression

-   -   (A) Human ALL-SCID6 and ALL-SCID3, ALL-SCID7 and pre-B-ALL-SCID        derived from xenografted mice display different levels of        opioid-receptors on their cell surface. ALL-SCID6, ALL-SCID3 and        ALL-SCID7 were stained with naloxone-fluoresceine measuring        opioid-receptor expression (OR, thick black curve) and analyzed        by flowcytometry. Controls (Co) are exhibited as thin black        curves.    -   (B) ALL-SCID6, ALL-SCID3, ALL-SCID7 and pre-B-ALL-SCID were        treated with different concentrations of D,L-methadone (as        indicated). After 24h and 48h, the percentages of apoptotic        cells were measured by FSC/SSC-analysis. The percentage of        specific apoptosis was calculated as follows: 100×[experimental        dead cells (%)−spontaneous dead cells in medium        (%)]/[100%−spontaneous dead cells in medium(%)]. Columns, mean        of triplicates; bars, SD<10%.

FIG. 2: Combination treatment with D,L-methadone and doxorubicin inducesapoptosis in ALL-cells expressing moderate amounts of opioid receptors

-   -   (A) Different BCP-ALL-cell lines (Tanoue, Nalm6 and Reh) express        a moderate number of opioid-receptors on their cell surface.        Tanoue, Nalm6 and Reh were stained with naloxone-fluoresceine        measuring opioid-receptor expression (OR, thick black curve) and        analyzed by flowcytometry. Controls (Co) are exhibited as thin        black curves.    -   (B) BCP-ALL-cell lines (Tanoue, Nalm6 and Reh) were treated with        different concentrations of D,L-methadone (as indicated) alone        (−Doxo, white columns), with doxorubicin alone or with        D,L-methadone (as indicated) in addition to doxorubicin (+Doxo,        black columns). For the cell line Tanoue, we used doxorubicin in        a concentration of 0.06 μg/mL, for Nalm6 and Reh a concentration        of 0.01 μg/mL. 120h after stimulation, percentages of apoptotic        cells were measured by hypodiploid DNA analysis.    -   (C) D,L-methadone strongly enhances doxorubicin sensitivity of        xenograft-derived-BCP-ALL-patient-cells ex vivo.        Xenograft-derived-BCP-ALL-cells (pre-B-ALL-SCID) were treated        with different concentrations of D,L-methadone (as indicated)        alone (−Doxo, white columns) with 0.01 μg/mL doxorubicin alone        or with D,L-methadone in addition to doxorubicin (+Doxo, black        columns). 48h after stimulation, the percentages of apoptotic        cells were measured by FSC/SSC-analysis. The percentage of        specific apoptosis was calculated as described in FIG. 1B.        Columns, mean of triplicates; bars, SD<10%.

FIG. 3: D,L-methadone in combination with doxorubicin restores deficientactivation of apoptotic pathways in BCP-ALL-cells expressing moderateamounts of opioid receptors in vitro

-   -   (A) D,L-methadone and doxorubicin co-treatment provokes caspases        activation. The BCP-ALL-cell line Tanoue was treated with        D,L-methadone (as indicated) alone (−Doxo), with 0.06 μg/mL        doxorubicin (+Doxo) alone or with D,L-methadone (as indicated)        in addition to doxorubicin (+Doxo). After 120h Western blot        analyses for caspase-2, caspase-9, caspase-3 and PARP were        performed. Downregulation of procaspase-2 was detected at ˜48        kDa. The active fragment of caspase-9 was detected at ˜37 kDa,        the active fragment of caspase-3 at ˜19 kDa and ˜17 kDa and PARP        cleavage at ˜85 kDa. Equal protein loading was controlled by        anti-β-actin antibody.    -   (B) D,L-methadone and doxorubicin-induced apoptosis depends on        caspase activation. Pre-incubation of the cell line Tanoue with        50 μM of the caspase inhibitor zVAD.fmk for 1h (white columns)        or without pre-treatment (black columns) was followed by        addition of D,L-methadone (as indicated) in combination with        0.06 μg/mL doxorubicin. Apoptosis induction was detected 120h        after stimulation by FSC/SSC-analysis. The percentage of        specific apoptosis was calculated as described in FIG. 1B.        Columns, mean of triplicates; bars, SD<10%.    -   (C) Downregulation of XIAP and Bcl-x_(L) by D,L-methadone and        doxorubicin co-treatment. The cell line Tanoue was treated with        D,L-methadone (as indicated) alone (−Doxo), with 0.06 μg/mL        doxorubicin (+Doxo) alone or with D,L-methadone (as indicated)        in addition to doxorubicin (+Doxo). After 120h Western blot        analyses for XIAP and Bcl-x_(L) were performed. XIAP was        detected at 58 kDa and Bcl-x_(L) at ˜30 kDa. Equal protein        loading was controlled by anti-β-actin antibody.

FIG. 4: Doxorubicin enhances opioid receptor expression whereasD,L-methadone enhances doxorubicin uptake and inhibits its efflux

-   -   (A) Doxorubicin enhances opioid receptor expression on the        cells' surface. The BCP-ALL-cell line Tanoue was treated for 96h        with 0.06 μg/mL doxorubicin. After staining of        doxorubicin-treated (+Doxo) and untreated cells (−Doxo) with        naloxone-fluoresceine relative fluorescence intensities were        determined flowcytometrically. X-fold increase in opioid        receptor expression is shown after subtracting the cells'        autofluorescence (−Doxo) and doxorubicin fluorescence (+Doxo).    -   (B) D,L-methadone enhances doxorubicin uptake and inhibits its        efflux. The BCP-ALL-cell line Tanoue was either pre-treated with        0.3 μg/mL doxorubicin (Doxo) alone or with a combination of        doxorubicin and 10 μg/mL D,L-methadone (Doxo+methadone) for        24 h. Maximal doxorubicin cell uptake was analyzed via        doxorubicin fluorescence in cells using flowcytometry after 24 h        (Oh, max. uptake). After washing doxorubicin-treated cells,        cells were either left untreated (Doxo) or treated with 10 μg/mL        D,L-methadone (Doxo+Methadone) and incubated for different        points in time (8h, 24h). Doxorubicin efflux was analyzed via        doxorubicin fluorescence in cells using flowcytometry after 8h        and 24h. Values are mean fluorescence intensities+/−SE.

FIG. 5: Combination treatment with D,L-methadone and doxorubicin inducedapoptosis depends on opioid-receptor triggering via downregulation ofcAMP

-   -   (A) Inhibition of opioid-receptor triggering inhibits apoptosis        induction mediated by combination treatment with D,L-methadone        and doxorubicin. The BCP-ALL-cell line Tanoue was incubated with        60 μg/mL naloxone (Naloxone), 3 μg/mL D,L-methadone        (D,L-Methadone) and 0.06 μg/mL doxorubicin (Doxo) alone or in        different combinations as indicated. After 96 h, the percentages        of apoptotic cells were measured by FSC/SSC-analysis.    -   (B) Inhibition of opioid-receptor triggering inhibits caspase        activation mediated by combination treatment with D,L-methadone        and doxorubicin. The BCP-ALL-cell line Tanoue was incubated with        60 μg/mL naloxone (Naloxone), 3 μg/mL D,L-methadone        (D,L-Methadone) and 0.06 μg/mL doxorubicin (Doxo) alone or in        different combinations as indicated. Western blot analyses for        caspase-2, caspase-9, caspase-3 and PARP were performed after        96h of incubation. Downregulation of procaspase-2 was detected        at ˜48 kDa. The active fragment of caspase-9 was detected at ˜37        kDa, of caspase-3 at ˜19 kDa and ˜17 kDa and PARP cleavage at        ˜85 kDa. Equal protein loading was controlled by anti-β-actin        antibody.    -   (C) Increasing cAMP levels via repression of phosphodiesterase        activity inhibits apoptosis. The BCP-ALL-cell line Tanoue was        incubated for 96h with 200 μM 3-Isobutyl-1-methylxanthine        (IBMX), 3 μg/mL D,L-methadone (D,L-Methadone) and 0.06 μg/mL        doxorubicin (Doxo) alone or in different combinations as        indicated. (D) Uncoupling inhibitory G-proteins from opioid        receptors inhibits apoptosis by preventing inhibition of        adenylyl cyclase. The BCP-ALL-cell line Tanoue was incubated        with 20 ng/mL pertussis toxin (PTX), 3 μg/mL D,L-methadone        (D,L-Methadone) and 0.06 μg/mL doxorubicin (Doxo) alone or in        different combinations as indicated. After 96 h, the percentages        of apoptotic cells were measured by FSC/SSC-analysis. The        fraction of apoptotic cells were determined by FSC/SSC-analysis.        The percentage of specific apoptosis was calculated as described        in FIG. 1B. Columns, mean of triplicates; bars, SD<10%.

FIG. 6: D,L-methadone inhibits growth of leukemia xenografts andincreases doxorubicin sensitivity Fragments of an in vivo passage of apatient-derived T-ALL (ALL-SCID6, see also FIG. 1) were transplantedinto male NSG mice. Mice were treated with D,L-methadone alone (n=8,orally day 1-76, D,L-Methadone), with doxorubicin alone (n=8, i.v. day46, 53, 60, 76, Doxorubicin) or with a combination treatment withD,L-methadone and doxorubicin (n=8, D,L-Methadone+Doxo). D,L-methadonewas used in weekly increasing doses from 20 up to 120 mg/kg/day anddoxorubicin in a dose of 3 mg/kg. As control group xenografted mice weretreated i.v. with 10% Tween 80 in saline (n=8, Vehicle). For 76 daysafter transplantation all mice were monitored for tumour growth, bodyweight and health condition. *significant to vehicle (p<0.05,Mann-Whitney U test).

FIG. 7: Opioid receptor signaling. Stimulation of opioid receptors (OR)by agonists like D,L-methadone leads to an activation of the inhibitoryG_(i)-protein. The α_(i)-subunit inactivates adenylyl cyclase (AC)resulting in a reduction of cAMP levels within the cell which in turnleads to apoptosis which might be mediated by several differentmodulators. Also the βγ-subunits of the G_(i)-protein modulate theactivity of different effectors like the inhibition of Ca²⁺- and theactivation of K⁺-channels.

FIG. 8: Opioid receptor expression on glioblastoma cells. Theglioblastoma cell lines U118MG and A172 were stained with naloxonefluorescein measuring opioid receptor expression (OR, thick black curve)and analysed by flow cytometry. Controls (Co) are exhibited as thinblack curves.

FIG. 9: D,L-methadone sensitizes glioblastoma cells for doxorubicintreatment. Glioblastoma cells A172 were incubated with 3 μg/mLD,L-methadone alone, with 0.1 μg/mL doxorubicin (0.1 μg/mL Doxo) aloneor with 3 μg/mL D,L-methadone in combination with 0.1 μg/mL doxorubicin(Doxo+D,L-Methadone). Control represents untreated glioblastoma cells.After 144h light microscopy pictures were taken. Cotreatment of A172with 3 μg/mL D,L-methadone and 0.1 μg/mL doxorubicin led to detachmentof the cells from the ground, membrane-blebbing and cell-shrinkage.

FIG. 10: Combination treatment with D,L-methadone and doxorubicininduces apoptosis in glioblastoma cells. A172 and U118MG glioblastomacells were treated with different concentrations of D,L-methadone (10,3, 1 μg/mL) alone (Medium, white columns), with doxorubicin (0.1 μg/mLDoxo, black columns) alone or with different concentrations ofD,L-methadone (10, 3, 1 μg/mL) in addition to doxorubicin (0.1 μg/mLDoxo, black columns). After 120h and 144h the percentages of apoptoticcells were measured by hypodiploid DNA analysis. The percentage ofspecific apoptosis was calculated as follows: 100×[experimental deadcells (%)−spontaneous dead cells in medium (%)]/[100%−spontaneous deadcells in medium (%)]. Columns, mean of triplicates; bars, SD<10%.Similar results were obtained in three independent experiments.

FIG. 11: D,L-methadone sensitizes primary human glioblastoma cells fordoxorubicin treatment. (A) Primary human glioblastoma cells were stainedwith naloxone fluorescein measuring opioid receptor expression (OR,thick black curve) and analyzed by flow cytometry. Control (Co) isexhibited as thin black curve. (B) Primary human glioblastoma cells weretreated with different concentrations of D,L-methadone (3, 1 μg/mL)alone (Medium, white columns) with 0.1 μg/mL doxorubicin (0.1 μg/mLDoxo, black columns) alone or with D,L-methadone (3, 1 μg/mL) inaddition to 0.1 μg/mL doxorubicin (0.1 μg/mL Doxo, black columns). After120h the percentages of apoptotic cells were measured by hypodiploid DNAanalysis. The percentage of specific apoptosis was calculated asdescribed in FIG. 1 c. Columns, mean of triplicates, bars, SD<10%.Similar results were obtained in three independent experiments

FIG. 12: D,L-methadone sensitizes glioblastoma-initiating stem cells fordoxorubicin treatment. (A) Glioblastoma-initiating stem cells werestained with naloxone fluorescein measuring opioid receptor expression(OR, thick black curve) and analyzed by flow cytometry. Control (Co) isexhibited as thin black curve. (B) Glioblastoma-initiating-stem cellswere treated with different concentrations of D,L-methadone (10, 3, 1μg/mL) alone (Medium, white columns), with 0.1 μg/mL doxorubicin (0.1μg/mL Doxo, black columns) alone or with D,L-methadone (3, 1 μg/mL) inaddition to 0.1 μg/mL doxorubicin (0.1 μg/mL Doxo, black columns). After144h the percentages of apoptotic cells were measured by hypodiploid DNAanalysis. The percentage of specific apoptosis was calculated asdescribed in FIG. 1 c. Columns, mean of triplicates; bars, SD<10%.Similar results were obtained in three independent experiments.

FIG. 13: Cell death induction of glioblastoma cells using D,L-methadoneand doxorubicin cotreatment depends on caspases activation. (A)D,L-methadone restored deficient caspases activation by doxorubicin inglioblastoma cells. A172 were treated with different concentrations ofD,L-methadone (3, 1 μg/mL) alone, with 0.1 μg/mL doxorubicin (0.1 μg/mLDoxo) alone or with different concentrations of D,L-methadone (3, 1μg/mL) in addition to doxorubicin (0.1 μg/mL Doxo). After 144h Westernblot analyses for caspase-10, -2, -9, -3 and PARP were performed.Downregulation of procaspase-10 was detected at ˜58 kDa and ofprocaspase-2 at ˜48 kDa. The active fragment of caspase-9 was detectedat ˜37 kDa, the active fragment of caspase-3 at ˜17 kDa and PARPcleavage at ˜85 kDa. Equal protein loading was controlled byanti-β-actin antibody. (B) Inhibition of caspases activation with thebroad spectrum inhibitor of caspases zVAD.fmk blocks apoptosis inducedby cotreatment of D,L-methadone and doxorubicin in A172 cells.Glioblastoma cells A172 were treated with different concentrations ofD,L-methadone (10, 3, 1 μg/mL) in combination with 0.1 μg/mL doxorubicin(+0.1 μg/mL Doxo) in the absence (Medium, black columns) or presence of50 μmol/L of zVAD.fmk (white columns, 50 μmol/L zVAD.fmk). After 120hand 144h, the percentages of apoptotic cells were measured byhypodiploid DNA analysis. The percentage of specific apoptosis wascalculated as described in FIG. 1 c. Columns, mean of triplicates; bars,SD<10%. Similar results were obtained in three independent experiments.(C) Downregulation of XIAP and Bcl-x_(L) in glioblastoma cells by usingD,L-methadone in combination with doxorubicin. Glioblastoma cells A172were treated with different concentrations of D,L-methadone (3, 1 μg/mL)alone, with 0.1 μg/mL doxorubicin (0.1 μg/mL Doxo) alone or withD,L-methadone (3, 1 μg/mL) in addition to doxorubicin (0.1 μg/mL Doxo)for 144 h. Western blot analyses for XIAP and Bcl-x_(L) were performed.XIAP was detected at 58 kDa and Bcl-x_(L) was detected at 30 kDa. Equalprotein loading was controlled by anti-β-actin antibody.

FIG. 14: D,L-methadone reversed deficient activation of apoptosispathways by doxorubicin in glioblastoma-initiating-stem cells. (A)Glioblastoma-initiating-stem cells were treated with D,L-methadone (3μg/mL) alone, with 0.1 μg/mL doxorubicin (0.1 μg/mL Doxo) alone or withD,L-methadone (3 μg/mL) in addition to doxorubicin (0.1 μg/mL Doxo).After 144h Western blot analyses for caspase-10, -2, -9, -3 and PARPwere performed. Downregulation of procaspase-10 was detected at ˜58 kDaand of procaspase-2 at ˜48 kDa. The active fragment of caspase-9 wasdetected at ˜37 kDa, the active fragment of caspase-3 at ˜19 kDa and ˜17kDa and PARP cleavage at ˜85 kDa. Equal protein loading was controlledby anti-β-actin antibody. (B) Glioblastoma-initiating-stem cells weretreated with D,L-methadone (3 μg/mL) alone, with 0.1 μg/mL doxorubicin(0.1 μg/mL Doxo) alone or with D,L-methadone (3 μg/mL) in addition todoxorubicin (0.1 μg/mL Doxo) for 144h. Western blot analyses for XIAP,Bcl-x_(L) and Bcl-x_(s) were performed. XIAP was detected at 58 kDa,Bcl-x_(L) was detected at 30 kDa and Bcl-x_(S) was detected at 27 kDa.Equal protein loading was controlled by anti-β-actin antibody.

FIG. 15: D,L-methadone enhances doxorubicin uptake and inhibits itsefflux. (A) D,L-methadone enhances doxorubicin-accumulation in theglioblastoma cell line A172. A172 were incubated with 0.3 μg/mLdoxorubicin alone or in combination with 10 μg/mL D,L-methadone. After4, 8 and 24h incubation the fluorescence intensity of doxorubicin (Doxo)using flow cytometry analysis were determined. In the graphic therelative doxorubicin-uptake is shown. Columns, mean of triplicates;bars, SD <10%. Similar results were obtained in three independentexperiments. (B) A172 were incubated with 0.3 μg/mL doxorubicin for 4h.At distinct points in time (4, 8 and 24h) after washing thedoxorubicin-containing medium away (Oh) the fluorescence intensity ofdoxorubicin using flow cytometry analysis was determined. In the graphicthe relative doxorubicin-content is shown. Columns, mean of triplicates;bars, SD <10%. Similar results were obtained in three independentexperiments.

FIG. 16: 1 Doxorubicin enhances opioid receptor expression on the cellsurface. (A) The glioblastoma cell line A172 was treated for 106 h with0.1 μg/mL doxorubicin. After staining of doxorubicin-treated(doxorubicin) and untreated cells with naloxone-fluorescein (naloxone)relative fluorescence intensities were determined flowcytometrically.(B) Tabular summary of untreated and doxorubicin control cells, naloxonetreated cells, whereas D_((naloxone-control)) represents the medianfluorescence intensities after subtracting the cells' autofluorescence(control).

FIG. 17: D,L-methadone sensitizes leukemia cancer cells (Nalm-6),pancreatic cancer cells (Colo357) and ovarian cancer cells (A2780) foretoposide or cisplatin treatment. The Nalm6, Colo357 and A2780 cellswere treated with different concentrations of D,L-methadone (10, 3, 1μg/mL) alone (Medium, white columns), with 0.03 μg/mL Etoposide or 0.3μg/mL cisplatin alone or with D,L-methadone (10, 3, 1 μg/mL) in additionto 0.03 μg/mL Etoposide (0.03 μg/mL Etoposide, black columns) or 0.3μg/mL cisplatin (0.3 μg/mL cisplatin, black columns). After 120 to 144hthe percentages of apoptotic cells were measured by hypodiploid DNAanalysis. The percentage of specific apoptosis was calculated asdescribed in FIG. 1 c. Columns, mean of triplicates; bars, SD<10%.Similar results were obtained in three independent experiments.

FIG. 18: D,L-methadone sensitizes chronic lymphocytic leukemia (CLL)cells for Fludarabine treatment. The CLL cells were treated withdifferent concentrations of D,L-methadone (10, 3, 1 μg/mL) alone(Medium, white columns), with 0.1 μM Fludarabine (0.1 μM Fludarabine,black columns) alone or with D,L-methadone (30, 10, 5, 3, 1, 0.5, 0.3,0.1 μg/mL) in addition to 0.1 μM Fludarabine (0.1 μM Fludarabine, blackcolumns). After 72h the percentages of apoptotic cells were measured byhypodiploid DNA analysis. The percentage of specific apoptosis wascalculated as described in FIG. 1B. Columns, mean of triplicates; bars,SD<10%. Similar results were obtained in three independent experiments.

FIG. 19: Cisplatin enhances opioid receptor expression in HL60 cellsCisplatin enhances opioid receptor expression on the surface of thepromyelocytic leukemia cell line HL60. The HL60 cell line was treatedfor 24h with 0.3 μg/mL cisplatin. After staining of cisplatin-treated(+Cisplatin) and untreated cells (−Cisplatin) with naloxone-fluoresceinerelative fluorescence intensities were determined flowcytometrically.2.1-fold increase in opioid receptor expression is shown aftersubtracting the cells autofluorescence.

FIG. 20: The Her2/neu-resistent mamma carcinoma cell line JIMT-1expresses high levels of the p-opioid receptor The human JIMT-1 cellline, derived from a pleural metastasis of a 62-year old patient withbreast cancer who was clinically resistant to Herceptin displaysopioid-receptors on its cell surface. JIMT-1 cells were stained withnaloxone-fluorescein measuring opioid-receptor expression (OR, thickblack curve) and analyzed by flow cytometry. Controls (Co) are exhibitedas thin black curves.

FIG. 21: Cell cycle analysis and apoptosis of the Her2/neu-resistentmamma carcinoma cell line JIMT-1 treated with a combination ofD,L-methadone and doxorubicin. The human JIMT-1 cell line was treatedwith 1, 3 or 10 μg/mL of methadone alone (1, 3, 10 Met −Doxo), with0.015 μg/mL of doxorubicin (Doxo) or a combination of 0.015 μg/mL ofdoxorubicin with 1, 3 or 10 μg/mL of methadone (1,3,10 Met+Doxo). (A) AFACS analysis of the cells revealed that the combination of bothsubstances dose-dependently increased apoptosis of the JIMT-1 cells at96 hours after treatment. (B) A FACS analysis performed at 96 hours(left side of the figure) and 120 hours (right side of the figure) aftertreatment showed further increased levels of apoptosis due tocombination treatment. 0 or 0,015 μg/ml doxorubicin (black bars), 1μg/mL methadone plus 0 or 0,015 μg/ml doxorubicin (white bars), 3 μg/mLmethadone plus 0 or 0,015 μg/ml doxorubicin (hatched bars), 10 μg/mLmethadone plus 0 or 0,015 μg/ml doxorubicin (doted bars).

FIG. 22: Cell death induction of JIMT-1 cells using D,L-methadone anddoxorubicin cotreatment depends on caspases activation. Inhibition ofcaspase activation with the broad spectrum caspase inhibitor zVAD.fmkblocks apoptosis induced by cotreatment of D,L-methadone and doxorubicinin JIMT-1 cells. The human cell line JIMT-1 was treated with differentconcentrations of D,L-methadone (10, 3, 1 μg/mL) in combination with0.015 μg/mL doxorubicin (+0.015 μg/mL Doxo) in the absence (left sideddiagrams) or presence of 50 μmol/L of zVAD.fmk (diagrams on the rightside). At 96 hours (A) or 120 hours (B) after addition of the drugcombination, the cells were analysed using flow cytometry.

FIG. 23: Cell death induction of JIMT-1 cells using D,L-methadone anddoxorubicin cotreatment depends on caspases activation. (A)D,L-methadone restored deficient caspases activation by doxorubicin inJIMT-1. A172 were treated with different concentrations of D,L-methadone(10, 3, 1 μg/mL) alone, with 0.015 μg/mL doxorubicin alone or withdifferent concentrations of D,L-methadone (10, 3, 1 μg/mL) in additionto 0.015 μg/ml doxorubicin. After 96h Western blot analyses forcaspase--8, -9, -3 and PARP were performed. The active fragment ofcaspase-8 was detected at −43 kDa, the active fragment of caspase-9 wasdetected at ˜37 kDa, the active fragment of caspase-3 at ˜17 kDa andPARP cleavage at ˜85 kDa. Equal protein loading was controlled byanti-β-actin antibody. (B) Downregulation of XIAP and Bcl-x_(L) inJIMT-1 cells by using D,L-methadone in combination with doxorubicin.Mamma carcinoma cells JIMT-1 were treated with different concentrationsof D,L-methadone (10, 3, 1 μg/mL) alone, with 0.015 μg/mL doxorubicinalone or with D,L-methadone (10, 3, 1 μg/mL) in addition to doxorubicin(0.015 μg/mL Doxo) for 96h. Western blot analyses for XIAP and Bcl-x_(L)were performed. XIAP was detected at 57 kDa and Bcl-x_(L) was detectedat 21 kDa. Equal protein loading was controlled by anti-β-actinantibody.

FIG. 24: Induction of apoptosis in T-Cell leukemia CEM cells by acombination of doxorubicin and fentanyl. Human T-Cell leukemia CEM cellline (10000 cells/100 μl) were treated with 30, 10, 5, 3, 1, 0.5, 0.3,0.1 μg/mL fentanyl alone (white bars) or in addition to 0.02 μg/mLdoxorubicin (black bars). After 48h and 72h quantification of apoptosiswas measured by flow cytometry.

FIG. 25: Induction of apoptosis in human acute myeloid leukemia HL-60cells by a combination of doxorubicin and buprenorphine. Human acutemyeloid leukemia HL-60 cell line (5000 cells/100 μl) were treated with20, 10, 5, 3, 1, 0.5, 0.3, 0.1 μg/mL buprenorphine alone (white bars) orin addition of 0.003 μg/mL doxorubicin (black bars). After 144h or 168hquantification of apoptosis was measured by flow cytometry.

FIG. 26: Schematic diagram showing the mutual positive interactionbetween opioids and anticancer drugs. On one side, opioids enhance thecellular uptake and inhibit the efflux of anticancer drugs. On the othersides anticancer drugs lead to an increased expression of opioidreceptors. Hence, both agents can exert their cytotoxic potential to ahigher extent.

FIG. 27: Opioid receptor expression on different leukemia

Different leukemia cells (human T cell leukemia, human acute myeloidleukemia, human B cell precursor leukemia and human B cell leukemia)express different moderate number of opioid-receptors on their cellsurface. Leukemia cells were stained with naloxone-fluoresceinemeasuring opioid-receptor expression (OR, thick black curve) andanalyzed by flowcytometry. Controls (Co) without naloxone are exhibitedas thin black curves.

FIG. 28: Effect of combination therapy of opioid receptor agonist andanticancer agent

D,L-methadone strongly enhances cisplatin sensitivity of differentleukemia cells. Different leukemia cells (human T cell leukemia, humanacute myeloid leukemia, human B cell precursor leukemia and human B cellleukemia) were treated with different concentrations of D,L-methadone(as indicated) alone (−CDDP, white columns) with cisplatin alone or withD,L-methadone in addition to cisplatin (+CDDP, black columns). Aftertime of incubation, the percentages of apoptotic cells were measured byFSC/SSC-analysis. The percentage of specific apoptosis was calculated asdescribed in FIG. 1 B. Columns, mean of triplicates; bars, SD<10%.

FIG. 29: D,L-methadone in combination with cisplatin restores deficientactivation of apoptotic pathways in leukemia cells. D,L-methadone andcisplatin co-treatment provokes caspases activation and inducesdownregulation and cleavage of XIAP and upregulation of Bax. Differentleukemia cells (human T cell leukemia, human acute myeloid leukemia,human B cell precursor leukemia) were treated with D,L-methadone (asindicated) alone (−CDDP), with cisplatin (CDDP) alone or withD,L-methadone (as indicated) in addition to cisplatin (+CDDP). Aftertime of incubation Western blot analyses for caspase-2, caspase-9,caspase-3, PARP, XIAP and Bax were performed. Downregulation ofprocaspase-2 was detected at ˜48 kDa. The active fragment of caspase-9was detected at ˜37 kDa, the active fragment of caspase-3 at ˜19 kDa andor ˜17 kDa, PARP at ˜116 kDa, PARP cleavage at ˜85 kDa, XIAP wasdetected at ˜58 kDa and XIAP cleavage at ˜30 kDa and Bax at ˜21 kDa.Equal protein loading was controlled by anti-β-actin antibody.

FIG. 30: D,L-methadone and cisplatin-induced apoptosis depends oncaspase activation. Pre-incubation of different leukemia cells (human Tcell leukemia, human acute myeloid leukemia, human B cell precursorleukemia) with 50 μM of the caspase inhibitor zVAD.fmk for 1h(+zVAD.fmk, white columns) or without pre-treatment (−zVAD.fmk, blackcolumns) was followed by addition of D,L-methadone (as indicated) incombination with cisplatin (as indicated). Apoptosis induction wasdetected after time of incubation by FSC/SSC-analysis. The percentage ofspecific apoptosis was calculated as described in FIG. 1B. Columns, meanof triplicates; bars, SD<10%.

FIG. 31: Cisplatin enhances opioid receptor expression. (A) Cisplatinenhances opioid receptor expression on the cells' surface of differentleukemia cells (human T cell leukemia, human acute myeloid leukemia andhuman B cell precursor leukemia) were treated with cisplatin (asindicated). After staining of cisplatin-treated cells (CDDP) anduntreated cells (Co) with naloxone-fluoresceine relative fluorescenceintensities were determined flowcytometrically. X-fold increase inopioid receptor expression compared to the untreated control group isshown after subtracting the cells' autofluorescence and cisplatinfluorescence.

FIG. 32: Combination treatment with D,L-methadone and doxorubicininduced apoptosis depends on opioid-receptor triggering. Inhibition ofopioid-receptor triggering inhibits apoptosis induction mediated bycombination treatment with D,L-methadone and doxorubicin. Glioblastomacells were incubated with 60 μg/mL naloxone (Naloxone), 3 μg/mLD,L-methadone (D,L-Methadone) and 0.1 μg/mL doxorubicin (Doxo) alone orin different combinations as indicated by the marks+ and − . . . After120h and 144h, the percentages of apoptotic cells were measured byFSC/SSC-analysis. The results of the different treatments which areindicated concerning the given substances under the single bars after120 h (left side of the figure) and 144 h (right side of the figure) areshown in FIG. 32.

FIG. 33: Different duration of effectiveness of different opioidsinduces different rates of cell death in glioblastoma cells.

-   -   A) Glioblastoma cells were treated with different concentrations        of D,L-methadone (as indicated) alone (−Doxo, white columns,        which are very low and on the left side of a black bar), with        doxorubicin alone or with D,L-methadone (as indicated) in        addition to doxorubicin (+Doxo, black columns) using the same        concentration of 0,1 μg/mL doxorubicin for all treatments and        different concentrations of D,L-methadone as indicated. 144h        after stimulation, percentages of cell death and apoptotic cells        were measured by hypodiploid DNA analysis.    -   B) Glioblastoma cells were treated with different concentrations        of morphine (as indicated) alone (−Doxo, white columns which are        very low and on the left side of a black bar), with doxorubicin        alone or with morphine (as indicated) in addition to doxorubicin        (+Doxo, black columns) using the same concentration of 0,1 μg/mL        doxorubicin for all treatments and different concentrations of        D,L-methadone as indicated. 144 h after stimulation, percentages        of cell death and apoptotic cells were measured by hypodiploid        DNA analysis.    -   The percentage of specific cell death was calculated as        described in FIG. 1B. Columns, mean of triplicates; bars,        SD<10%.

FIG. 34: Different Duration of Effectiveness of Different OpioidsInduces Different Rates of Cell Death in Leukemia Cells

-   -   A) Leukemia cells (human B cell leukemia) were treated with        different concentrations of D,L-methadone (as indicated) alone        (−Doxo, white columns on the left side of a black bar), with        doxorubicin alone or with D,L-methadone (as indicated) in        addition to doxorubicin (+Doxo, black columns)) using the same        concentration of 0,1 μg/mL doxorubicin for all treatments and        different concentrations of D,L-methadone as indicated. 96h        after stimulation, percentages of apoptotic cells were measured        by hypodiploid DNA analysis.    -   B) Leukemia cells (human B cell leukemia) were treated with        different concentrations of morphine (as indicated) alone        (−Doxo, white columns on the left side of a black bar), with        doxorubicin alone or with morphine (as indicated) in addition to        doxorubicin (+Doxo, black columns) using the same concentration        of 0,1 μg/mL doxorubicin for all treatments and different        concentrations of D,L-methadone as indicated. For the 96h after        stimulation, percentages of apoptotic cells were measured by        hypodiploid DNA analysis.    -   The percentage of specific apoptosis was calculated as described        in FIG. 1B. Columns, mean of triplicates; bars, SD<10%.

FIG. 35: Combination treatment with D,L-methadone and doxorubicininhibits proliferation and induces S/G2-M cell cycle arrest inglioblastoma cells. Flow cytometric analysis of glioblastoma cellstreated with methadone and doxorubicin was shown. Flow cytometricanalysis of untreated cells (Untreated cells) (G1 peak is higher than G2peak), cells treated with 1 μg/ml methadone (Methadone) (G1 peak ishigher than G2 peak) and cells treated with methadone in addition to 0,1μg/ml doxorubicin (Methadone+Doxo). Arrest of cell cycle progression atthe G2/M phase (G1 peak lower than in untreated cells and G2 peak ishigher than in untreated cells) was shown in glioblastoma cells treatedwith methadone in addition to doxorubicin after 96h (A). subG1 peak infront of G1 is the fragmentated DNA (percentage of cell death). Resultsare representative of 3 independent experiments.

FIG. 36: Apoptosis induction and caspase activation depend on opioidreceptor activation inducing cAMP downregulation in glioblastoma. Opioidreceptor activation triggering downregulation of cAMP plays a criticalrole in sensitizing glioblastoma cells for doxorubicin treatment. (A, B)Blocking opioid receptor activation inhibits apoptosis induction.Glioblastoma cell line A172 was incubated with 100 μg/mL naloxone(Naloxone), 3 μg/mL D,L-methadone (D,L-Methadone) and 0.3 μg/mLdoxorubicin (Doxo) alone or in different combinations as indicated.After (A) 120h and (B) 144h, the percentages of apoptotic cells weremeasured by hypodiploid DNA analysis. (C) Inhibition of opioidreceptor-activation inhibits caspase activation. A172 cells wereincubated with 100 μg/mL naloxone (Naloxone), 3 μg/mL D,L-methadone(D,L-Methadone) and 0.3 μg/mL doxorubicin (Doxo) alone or in differentcombinations as indicated. Western blot analyses for caspase-9,caspase-3 and PARP were performed after 120h of incubation. The activefragment of caspase-9 was detected at ˜37 kDa, of caspase-3 at ˜19 kDaand ˜17 kDa and PARP cleavage at ˜85 kDa. Equal protein loading wascontrolled by anti-β-actin antibody. (D) Increasing cAMP levels viarepression of phosphodiesterase activity inhibits apoptosis. A172 cellswere incubated for 120h with 25 μM 3-lsobutyl-1-methylxanthine (IBMX), 3μg/mL D,L-methadone (D,L-Methadone) and 0.3 μg/mL doxorubicin (Doxo)alone or in different combinations as indicated. After 120h, thepercentages of apoptotic cells were measured by hypodiploid DNAanalysis.

FIG. 37: D,L-Methadone inhibits tumour growth of glioblastoma. Theglioblastoma cell line U87MG (U87) was transplanted into nude-mice. Micewere daily treated with D,L-methadone (n=8, black square) or the vehicle10% Tween 80 in saline (n=8, black diamond). After transplantation micewere observed for 33 days. At distinct points in time (as indicated),tumour growth and tumour volume were measured. *Significant to control(p<0.05, Mann-Whitney U test).

FIG. 38: D,L-methadone sensitizes ovarian cancer cells for short-termcisplatin treatment. A2780 ovarian cancer cells were treated withdifferent concentrations of D,L-methadone (3, 1, 0 μg/mL) alone, with 5or 3 μg/mL cisplatin alone or in combination with D,L-methadone and 5μg/mL cisplatin (+5 μg/ml CDDP, black columns) or 3 μg/mL cisplatin (+3μg/mL, white columns). After 24h, 48h and 72h, the percentages of celldeath/apoptotic cells were measured.

FIG. 39: D,L-methadone sensitizes ovarian cancer cells for long-termcisplatin treatment. A2780 ovarian cancer cells were treated withdifferent concentrations of D,L-methadone (10, 3, 1, 0 μg/mL) alone,with cisplatin (2, 1, 0.5, 0.3 μg/mL) alone or in combination withD,L-methadone and cisplatin. After 72h, 96h, 120h and 144h, thepercentages of cell death/apoptotic cells were measured.

FIG. 40: D,L-methadone sensitizes cisplatin-resistant ovarian cancercells for cisplatin treatment. A2780cis ovarian cancer cells weretreated with different concentrations of D,L-methadone (10, 3, 1, 0μg/mL) alone, with cisplatin (3, 2, 1 μg/mL) alone or in combinationwith D,L-methadone and cisplatin. After 72h 96h, 120h and 144h, thepercentages of cell death/apoptotic cells were measured.

FIG. 41: D,L-methadone strongly sensitizes ovarian cancer cells forcisplatin treatment. A2780 ovarian cancer cells were treated withdifferent concentrations of D,L-methadone (10, 3, 1, 0 μg/mL) alone,with 2 μg/mL cisplatin alone (black column) and with 0.5 or 0.2 μg/mLcisplatin (hatched and grey columns, respectively) in combination withD,L-methadone as indicated. After 144h, the percentages of celldeath/apoptotic cells were measured.

FIG. 42: Opioid receptor activation using D,L-methadone sensitizesovarian cancer cells for cisplatin-induced activation of caspases.D,L-Methadone restored deficient caspases activation by cisplatin inovarian cancer cells. A2780 ovarian cancer cells were treated withdifferent concentrations of D,L-methadone (3, 1 μg/mL, −CDDP) alone,with 5 or 3 μg/mL cisplatin alone or with D,L-methadone (3, 1 μg/mL) inaddition to 5 or 3 μg/mL cisplatin (+CDDP). After 24h, Western blotanalyses were performed. The active fragment of caspase-9 was detectedat ˜37 kDa, the active fragment of caspase-3 at ˜19 and 17 kDa, activefragment of caspase-8 was detected at ˜43 and 41 kDa and PARP cleavageat ˜85 kDa. Equal protein loading was controlled by anti-β-actinantibody.

FIG. 43: Opioid receptor activation using D,L-methadone plays a criticalrole in sensitizing breast cancer cells for doxorubicin treatment.Blocking opioid receptor activation using opioid antagonist naloxonestrongly inhibits apoptosis induction induced by combination treatmentwith D,L-methadone and doxorubicin. Breast cancer cells JIMT-1 incubatedwith 100 μg/mL naloxone (Naloxone), 10 μg/mL D,L-methadone(D,L-Methadone) and 0.01 μg/mL doxorubicin (Doxo) alone or in differentcombinations as indicated. After 96h, the percentages of celldeath/apoptotic cells were measured.

FIG. 44: D,L-methadone sensitizes prostate cancer cells for cisplatintreatment. Prostate cancer cells PC-3 were treated with differentconcentrations of D,L-methadone (3, 1 μg/mL) alone, with 5 or 3 μg/mLcisplatin alone or in combination with D,L-methadone and 5 μg/mLcisplatin (+5 μg/ml CDDP, black columns) or 3 μg/mL cisplatin (+3 μg/mL,white columns). After 96h and 120h, the percentages of apoptotic cellswere measured by hypodiploid DNA analysis.

FIG. 45: D,L-methadone sensitizes leukemia cells for treatment withdifferent anticancer drugs. Leukemia cells Nalm6 were treated withD,L-methadone (3 μg/mL) alone, with different anticancer drugs alone asindicated (white columns) or in combination with D,L-methadone anddifferent anticancer drugs (black columns). After 96h, the percentagesof cell death/apoptosis were measured.

FIG. 46: D,L-methadone sensitizes glioblastoma cells for treatment withdifferent anticancer drugs from the same anti-cancer drug class.Glioblastoma cells A172 were treated with D,L-methadone (3 μg/mL) alone,with different anticancer drugs alone as indicated (white columns) or incombination with D,L-methadone and different anticancer drugs (blackcolumns). After 120h, the percentages of cell death/apoptosis weremeasured.

FIG. 47 Opioid receptor expression on pancreatic cancer cells.Pancreatic cancer cells Colo357 were stained with naloxone fluoresceinmeasuring opioid receptor expression (OR, thick black curve) by flowcytometry. Controls (Co) are exhibited as thin black curves. A strongexpression of opioid receptors on the surface of pancreatic cancer wasfound.

FIG. 48: Opioids such as D,L-methadone increase anti-cancer drug-inducedcell death in pancreatic cancer cells. Pancreatic cancer cells Colo 357were treated with D,L-methadone (10, 3, 1, 0 μg/mL) alone, withoxaliplatin or cisplatin alone as indicated or with a combination ofD,L-methadone and (A) oxaliplatin or (B) cisplatin. After 120h (A) and144 (B), the percentages of cell death/apoptosis were measured.

FIG. 49: Opioid receptor activation using D,L-methadone sensitizespancreatic cancer cells for cisplatin- and oxaliplatin-inducedactivation of caspases. D,L-Methadone restored deficient caspasesactivation by (A) oxaliplatin or (B) cisplatin in pancreatic cancercells. Pancreatic cancer cells Colo 357 were treated with differentconcentrations of D,L-methadone (3, 1 μg/mL) alone, with (A) oxaliplatin(2, 3 μg/mL) alone, or (B) with cisplatin (0.5 or 0.7 μg/mL) alone orwith (A) D,L-methadone (3, 1 μg/mL) in addition to 2 or 3 μg/mLoxaliplatin or with (B) D,L-methadone (3, 1 μg/mL) in addition to 0.5 or0.7 μg/mL cisplatin. After 120h, Western blot analyses were performed.The active fragment of caspase-9 was detected at ˜46 kDa, the activefragment of caspase-3 at ˜19 and 17 kDa, and PARP cleavage at ˜85 kDa.The inhibitory protein of caspases XIAP was detected at ˜57 kDa Equalprotein loading was controlled by anti-β-actin antibody.

FIG. 50: Inhibition of caspases activation blocks opioid-sensitizedpancreatic cancer cells for oxaliplatin- or cisplatin-induced apoptosis.Pancreatic cancer cells Colo 357 were treated with differentconcentrations of D,L-methadone (10, 3, 1, 0 μg/mL) in combination with(A) 3 μg/mL oxaliplatin, (B) 2 μg/mL oxaliplatin, (C) 0.7 μg/mLcisplatin or (D) 0.5 μg/mL cisplatin without (−zVAD.fmk, black columns)or with addition of 50 μmol/L zVAD.fmk (+zVAD, white columns). After120h and 144h, the percentages of cell death/apoptotic cells weremeasured.

FIG. 51: Opioids using D,L-methadone sensitizes glioblastoma cells fortemozolomide treatment. Glioblastoma cells A172 were treated withdifferent concentrations of D,L-methadone 3, 1, 0 μg/mL) alone, withtemozolomide alone or in combination with D,L-methadone and temozolomide(black columns). After 120h, the percentages of apoptotic cells weremeasured.

FIG. 52: Opioids using D,L-methadone strongly sensitizes pancreaticcancer cells for oxaliplatin and cisplatin treatment.

-   -   (A) Pancreatic cancer cells (Colo 357) were treated with        different concentrations of D,L-methadone (10, 3, 1, 0 μg/mL)        alone, with 10 μg/mL oxaliplatin (black column), 3, 2 μg/ml        oxaliplatin alone and with 3 μg/mL oxaliplatin in combination        with D,L-methadone (hatched columns) or 2 μg/mL oxaliplatin in        combination with D,L-methadone (white columns) as indicated.        After 120h, the percentages of cell death/apoptotic cells were        measured.    -   (B) Pancreatic cancer cells (Colo 357) were treated with        different concentrations of D,L-methadone (10, 3, 1, 0 μg/mL)        alone, with 10 μg/mL cisplatin (black column), 0.7 or 0.5 μg/ml        cisplatin alone and with 0.7 μg/mL cisplatin in combination with        D,L-methadone (hatched columns) or 0.5 μg/mL cisplatin in        combination with D,L-methadone (white columns) as indicated.        After 144h, the percentages of cell death/apoptotic cells were        measured.

FIG. 53: Opioids using D,L-methadone strongly sensitizes leukemia cellsfor doxorubicin treatment.

-   -   (A) Leukemia cells (Nalm6) were treated with different        concentrations of D,L-methadone (10, 3, 1, 0 μg/mL) alone, with        0.1 μg/ml doxorubicin (black column) alone, with 0.01 μg/ml        doxorubicin alone or with a combination of 0.01 μg/mL        doxorubicin and D,L-methadone (hatched columns) as indicated.        After 120h, the percentages of cell death/apoptotic cells were        measured.    -   (B) Leukemia cells (Reh) were treated with different        concentrations of D,L-methadone (10, 3, 1, 0 μg/mL) alone, with        0.1 μg/ml doxorubicin (black column) alone, with 0.01 μg/ml        doxorubicin alone, or with a combination of 0.01 μg/mL        doxorubicin and D,L-methadone (hatched columns) as indicated.        After 120h, the percentages of cell death/apoptotic cells were        measured.    -   (C) Leukemia cells (Tanoue) were treated with different        concentrations of D,L-methadone (10, 3, 1, 0 μg/mL) alone, with        0.1 μg/ml doxorubicin (black column) alone, with 0.01 μg/ml        doxorubicin alone, or with a combination of 0.01 μg/mL        doxorubicin and D,L-methadone (hatched columns) as indicated.        After 120h, the percentages of cell death/apoptotic cells were        measured.

FIG. 54: Opioids using D,L-methadone strongly sensitizes breast cancercells for doxorubicin treatment.

-   -   (A) Breast cancer cells resistant to HER2-targeted therapies        (JIMT-1) were treated with different concentrations of        D,L-methadone (10, 3, 1, 0 μg/mL) alone, with 0.1 μg/ml        doxorubicin (black column) alone or with 0.015 μg/ml doxorubicin        alone and with 0.015 μg/mL doxorubicin in combination with        D,L-methadone (hatched columns) as indicated. After 120h, the        percentages of cell death/apoptotic cells were measured.

FIG. 55: Opioids using D,L-methadone strongly sensitizes glioblastomacells for doxorubicin treatment.

Glioblastoma cells (A172) were treated with different concentrations ofD,L-methadone (10, 3, 1, 0 μg/mL) alone, with 1 μg/ml doxorubicin (blackcolumn) alone, with 0.1 μg/ml doxorubicin alone or with a combination of0.1 μg/mL doxorubicin and D,L-methadone (hatched columns) as indicated.After 144h, the percentages of cell death/apoptotic cells were measured.

FIG. 56: Combination treatment of morphine and fentanyl shows a strongsynergistic effect for inducing cell death in leukemia cells. HL60leukemia cells were treated with fentanyl (3, 1 μg/mL) alone (A) ormorphine (3, 1 μg/mL) alone (A) or with a combination of fentanyl andmorphine (B) at concentrations as indicated. After 96h, 120h and 144h,the percentages of specific cell death/apoptotic cells were measured.

LITERATURE

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1-20. (canceled)
 21. A combination of an opioid receptor agonist and atleast one anticancer agent for use in the treatment of cancer, wherein(a) said opioid receptor agonist is administered to a patient in one ormore doses to establish a therapeutically effective plasma level for aperiod of at least one week, and (b) at least one anticancer agent thatis selected from the group consisting of chemotherapeutical agents,cytotoxic agents, cytostatic agents, immunotoxic agents andradiotherapy, and is administered to establish a period with atherapeutically effective plasma level, and (c) said periods of a) andb) overlap.
 22. The combination according claim 21, wherein saidanticancer agent and said opioid receptor agonist are administeredsimultaneously or successively.
 23. The combination according claim 21,wherein said opioid receptor agonist is capable of inhibiting cellproliferation and/or inducing cell death.
 24. The combination accordingto claim 21, wherein the patient has received a pre-treatment comprisingan anticancer agent.
 25. The combination according to claim 21, whereinthe administration period for the opioid receptor agonist providing atherapeutically relevant dose is at least two weeks.
 26. The combinationaccording to claim 21 wherein the opioid receptor agonist is selectedfrom the group consisting of: i. compounds of the methadone groupselected from the group consisting of D/L-methadone, D-methadone,L-methadone, and normethadone; ii. fentanyl derivatives selected fromthe group consisting of fentanyl, sufentanyl and carfentanyl; iii.morphinane compounds selected from the group consisting of morphine,codeine, heroine, dextrallorphane, dextromethorphan, dextrophanol,dimemorfan, levalorphan, levofurethylnormorphanol, levomethorphane,levophenacylmorphane, levorphanol, methorphane, morphanol, oxilorphan,phenomorphan, and xorphanol; iv. benzomorphane derivatives selected fromthe group consisting of 5,9-DEHB, alazocine, anazocine, bremazocine,butinazocine, carbazocine, cogazocine, cyclazocine, dezocine,eptazocine, etazocine, ethylketocyclazocine, fluorophen, gemazocine,ibazocine, ketazocine, metazocine, moxazocine, pentazocine, phenazocine,quadazocine, thiazocine, tonazocine, volazocine and 8-CAC; v.4-phenylpiperidine derivatives selected from the group consisting ofpethidine, ketobemidone, anileridine, piminodine, phenoperidine,furethidine, alpha-prodin, trimeperidine, 4-phenylpyrrolidinederivatives, profadol, 4-phenylazepanderivates, and meptazinol; vi.cyclohexane derivatives selected from the group consisting of tilidine,U-50488, tramadol and tapentadol; and vii. endogenous opioids selectedfrom the group consisting of endorphins, enkephalins, dynorphins,nociceptin, dermorphins, morphiceptin, endomorphines and fragmentsderived from the protein proopiomelanocortin (POMC).
 27. The combinationaccording to claim 21 wherein the opioid receptor agonist belongs to themethadone group.
 28. The combination according to claim 27, wherein theopioid receptor agonist is D/L methadone and the hydrochloride formthereof.
 29. The combination according to claim 21 wherein theanticancer agent is selected from the group consisting of: i.intercalating substances selected from the group consisting ofanthracycline, doxorubicin, idarubicin, epirubicin, and daunorubicin;ii. topoisomerase inhibitors selected from the group consisting ofirinotecan, topotecan, camptothecin, lamellarin D, etoposide,teniposide, mitoxantrone, amsacrine, ellipticines and aurintricarboxylicacid; iii. nitrosourea compounds selected from the group consisting ofcarmustine (BCNU), lomustine (CCNU), streptozocin; iv. nitrogen mustardsselected from the group consisting of cyclophosphamide, mechlorethamine,uramustine, bendamustine, melphalan, chlorambucil, mafosfamide,trofosfamid and ifosfamide; v. alkyl sulfonates selected from the groupconsisting of busulfan and treosulfan; vi. alkylating agents selectedfrom the group consisting of procarbazin, dacarbazin, temozolomid andthiotepa; vii. platinum analogues selected from the group consisting ofcisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, andtriplatin tetranitrate; viii. Microtubule disruptive drugs selected fromthe group consisting of vinblastine, colcemid and nocodazole; ix.antifolates selected from the group consisting of methotrexate,aminopterin, dichloromethotrexat, pemetrexed, raltitrexed andpralatrexate; x. purine analogues selected from the group consisting ofazathioprine, mercaptopurine, thioguanine, fludarabine, fludarabinephosphate, pentostatin and cladribine; xi. pyrimidine analogues selectedfrom the group consisting of 5-fluorouracil, floxuridine, cytarabine,6-azauracil, gemcitabine; xii. steroid hormones selected from the groupconsisting of gestagene, androgene, glucocorticoids, dexamethasone,prednisolone, and prednisone; xiii. anti-cancer antibodies selected fromthe group consisting of monoclonal antibodies, radioactively labeledantibodies and antibody-drug conjugates; xiv. anti-cancer peptidesselected from the group consisting of radioactively labeled peptides andpeptide-drug conjugates; xv. alpha, beta or gamma irradiation, electronparticles, or radioactively labeled chemical compounds; and xvi. Taxaneand Taxane analogues selected from the group consisting of paclitaxeland docetaxel.
 30. The combination according to claim 21 wherein theanticancer agent is methotrexate, cytarabine, cisplatin, temozolomide,etoposide, vincristine, thioguanine, gemcitabine, beta irradiation orgamma irradiation, and especially doxorubicin, cisplatin, oxaliplatin,rituximab or trastuzumab.
 31. The combination according to claim 21wherein patients suffer from a neoplasm as classified according theInternational statistical classification of Diseases and related healthproblems 10^(th) Revision (ICD-10), and wherein the neoplasm is selectedfrom the group consisting of malignant neoplasms of classes COO to C97,in situ neoplasms of classes D00 to D09, benign neoplasms of classes D10to D36, and neoplasms of uncertain or unknown behaviour of classes D37to D48.
 32. The combination according to claim 31 wherein patientssuffer from a neoplasm selected from the group consisting of pancreaticcarcinoma, hepatoblastoma, colon carcinoma, (small cell lung cancer,melanoma, mamma carcinoma, ovarian carcinoma, prostate carcinoma,glioblastoma, acute lymphoblastic leukemia, acute myeloid leukemia,chronic myeloid leukemia, chronic lymphocytic leukemia, pro-forms ofleukemia, hairy cell leukemia, Hodgkin's disease, Non-Hodgkin lymphoma,glioblastoma-initiating stem cells, tumour stem cells and multiplemyeloma.
 33. The combination according to claim 21, wherein the patientexhibits either an intrinsic or an acquired resistance.
 34. Thecombination according to claim 33, wherein the patient exhibits one ormore of the subsequent resistances: i. apoptosis resistance; ii.multi-drug resistance; iii. anticancer drug resistance; iv. cytotoxicdrug resistance; v. resistance to reactive oxygen species; vi.resistance to DNA damaging agents; vii. resistance to toxic antibodies;viii. doxorubicin or rituximab resistance; ix. cisplatin resistance; x.temozolomide resistance; xi. resistance to HER2 targeting therapy,selected from the group consisting of trastuzumab treatment xii. singleor cross resistance to one or more of the following drug substances:methotrexate, cytarabine, cisplatine, etoposide, vincristine,paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine,temozolomide, thioguanine, teniposide, dexamethasone, prednisolone,cyclophosphamide, diphosphamide, doxorubicin, epirubicin, idarubicin,daunorubicin, mercaptopurine and fludarabine; and xiii. Irradiationresistance.
 35. The combination according to claim 21, wherein theanticancer agent is given at a dose equal to or lower than therecommended dose for the treatment of cancer using the anticancer agentonly.
 36. The combination according to claim 35, wherein dose is 3 timeslower than the recommended dose for the treatment of cancer using theanticancer agent only.
 37. The combination according to claim 21,wherein at least one additional opioid receptor agonist is administeredto the patient.
 38. The combination according claim 37, wherein theopioid morphine is administered with the further opioid fentanyl to thepatient.
 39. A method for selection of the combination according toclaim 21 and/or the doses of the drugs used within said combinationcomprising the following steps: (a) providing an vitro culture of cancercells, cell lines or primary cells; (b) optionally testing the cellsfrom step (a) for expression of opioid receptors; (c) treating the cellsfrom step (a) with an opioid agonist, or at least one anticancer drug ora combination thereof; (d) analysing the cells for cell death/viabilityand/or expression of opioid receptors (e) selecting the opioidreceptor/anticancer drug combination and preferably a dose for saidcombination based on the desired extent of cell death/viability orinhibition of proliferation; and/or (f) selecting the anticancer agentand preferably a dose for said anticancer agent which shows the desiredextent of induction of opioid receptors.
 40. A method for selection ofan opioid receptor agonist for the treatment of cancer and a treatmentdose for said opioid receptor agonist comprising the following steps (a)providing an vitro culture of cancer cells, cell lines or primary cells;(b) optionally testing the cells from step (a) for expression of opioidreceptors; (c) treating the cells from step (a) with an opioid receptoragonist; (d) analysing the cells for cell death/viability; (e) selectingthe opioid receptor/anticancer drug combination and preferably a dosefor said combination based on the desired extent of cell death/viabilityor inhibition of proliferation; and/or (f) selecting the opioid receptoragonist and preferably a dose for said opioid receptor agonist whichshows the desired extent of induction of cell death.
 41. The combinationaccording to claim 21, wherein the administration period for the opioidreceptor agonist providing the therapeutically relevant dose is at leastfour weeks.
 42. The combination according to claim 21, wherein theadministration period for the opioid receptor agonist providing thetherapeutically relevant dose represents a chronic treatment.
 43. Thecombination according to claim 35, wherein the anticancer agent is givenat a dose 30 times lower than the recommended dose for the treatment ofcancer using the anticancer agent only.
 44. The combination according toclaim 35, wherein the anticancer agent is given at a dose 100 timeslower than the recommended dose for the treatment of cancer using theanticancer agent only.
 45. The method of claim 39, wherein said in vitroculture is isolated from a cancer biopsy or from a liquid sampleselected from the group consisting of blood, amniotic fluid, pleuralfluid, cerebrospinal fluid and peritoneal fluid.
 46. The method of claim40, wherein said in vitro culture is isolated from a cancer biopsy orfrom a liquid sample selected from the group consisting of blood,amniotic fluid, pleural fluid, cerebrospinal fluid and peritoneal fluid.